Gene Differentially Expressed in Breast and Bladder Cancer and Encoded Polypeptides

ABSTRACT

The present invention relates to a novel human gene that is differentially expressed in human carcinoma. More specifically, the present invention relates to methods of treating or preventing a disorder in a subject. The invention further relates to uses of C35 polypeptides in immunogenic compositions or vaccines, to induce antibody or T cell-mediated immunity against target cells, such as tumor cells, that express the C35 gene. The present invention further relates to use of C35 polypeptides in diagnosing a pathological condition or susceptibility to a pathological condition in a subject.

This application is a continuation application of U.S. application Ser.No. 11/601,669, filed Nov. 20, 2006, which is a divisional applicationof U.S. application Ser. No. 09/824,787, filed Apr. 4, 2001, whichclaims the benefit of U.S. Provisional Application No. 60/194,463, filedApr. 4, 2000, the entire contents of each are herein incorporated byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel human gene that isdifferentially expressed in human breast and bladder carcinoma. Morespecifically, the present invention relates to a polynucleotide encodinga novel human polypeptide named C35. This invention also relates to C35polypeptides, as well as vectors, host cells, antibodies directed to C35polypeptides, and the recombinant methods for producing the same. Thepresent invention further relates to diagnostic methods for detectingcarcinomas, including human breast and bladder carcinomas. The presentinvention further relates to the formulation and use of the C35 gene andpolypeptides in immunogenic compositions or vaccines, to induce antibodyand cell-mediated immunity against target cells, such as tumor cells,that express the C35 gene. The invention further relates to screeningmethods for identifying agonists and antagonists of C35 activity.

2. Background Art

Cancer afflicts approximately 1.2 million people in the United Stateseach year. About 50% of these cancers are curable with surgery,radiation therapy, and chemotherapy. Despite significant technicaladvances in these three types of treatments, each year more than 500,000people will die of cancer in the United States alone. (Jaffee, E. M.,Ann. N.Y. Acad. Sci. 886:67-72 (1999)). Because most recurrences are atdistant sites such as the liver, brain, bone, and lung, there is anurgent need for improved systemic therapies.

The goal of cancer treatment is to develop modalities that specificallytarget tumor cells, thereby avoiding unnecessary side effects to normaltissue. Immunotherapy has the potential to provide an alternativesystemic treatment for most types of cancer. The advantage ofimmunotherapy over radiation and chemotherapy is that it can actspecifically against the tumor without causing normal tissue damage. Oneform of immunotherapy, vaccines, is particularly attractive because theycan also provide for active immunization, which allows for amplificationof the immune response. In addition, vaccines can generate a memoryimmune response.

The possibility that altered features of a tumor cell are recognized bythe immune system as non-self and may induce protective immunity is thebasis for attempts to develop cancer vaccines. Whether or not this is aviable strategy depends on how the features of a transformed cell arealtered. Appreciation of the central role of mutation in tumortransformation gave rise to the hypothesis that tumor antigens arise asa result of random mutation in genetically unstable cells. Althoughrandom mutations might prove immunogenic, it would be predicted thatthese would induce specific immunity unique for each tumor. This wouldbe unfavorable for development of broadly effective tumor vaccines. Analternate hypothesis, however, is that a tumor antigen may arise as aresult of systematic and reproducible tissue specific gene deregulationthat is associated with the transformation process. This could give riseto qualitatively or quantitatively different expression of sharedantigens in certain types of tumors that might be suitable targets forimmunotherapy. Early results, demonstrating that the immunogenicity ofsome experimental tumors could be traced to random mutations (De Plaen,et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988); Srivastava, &Old, Immunol. Today 9:78 (1989)), clearly supported the firsthypothesis. There is, however, no a priori reason why random mutationand systematic gene deregulation could not both give rise to newimmunogenic expression in tumors. Indeed, more recent studies in bothexperimental tumors (Sahasrabudhe et al., J. Immunol. 151:6202-6310(1993); Torigoe et al., J. Immunol. 147:3251 (1991)) and human melanoma(van Der Bruggen et al., Science 254:1643-1647 (1991); Brichard et al.,J. Exp. Med. 178:489-495 (1993); Kawakami et al., Proc. Natl. Acad. Sci.USA 91:3515-3519 (1994); Boel et al., Immunity 2:167-175 (1995); Van denEynde et al., J. Exp. Med. 182: 689-698 (1995)) have clearlydemonstrated expression of shared tumor antigens encoded by deregulatednormal genes. The identification of MAGE-1 and other antigens common todifferent human melanoma holds great promise for the future developmentof multiple tumor vaccines.

In spite of the progress in melanoma, very few shared antigensrecognized by cytotoxic T cells have been described for other humantumors. The major challenge is technological. The most widespread and todate most successful approach to identify immunogenic molecules uniquelyexpressed in tumor cells is to screen a cDNA library with tumor-specificCTLs (cytotoxic T lymphocytes). Application of this strategy has led toidentification of several gene families expressed predominantly in humanmelanoma. Two major limitations of this approach, however, are that (1)screening requires labor intensive transfection of numerous small poolsof recombinant DNA into separate target populations, which themselvesoften need to be modified to express one or more MHC molecules requiredfor antigen presentation, in order to assay T cell stimulation by aminor component of some pool; and (2) with the possible exception ofrenal cell carcinoma, tumor-specific CTLs have been very difficult toisolate from either tumor infiltrating lymphocytes (TIL) or PBL ofpatients with other types of tumors, especially the epithelial cellcarcinomas that comprise greater than 80% of human tumors. It appearsthat there may be tissue specific properties that result intumor-specific CTLs being sequestered in melanoma.

Direct immunization with tumor-specific gene products may be essentialto elicit an immune response against some shared tumor antigens. It hasbeen argued that, if a tumor expressed strong antigens, it should havebeen eradicated prior to clinical manifestation. Perhaps then, tumorsexpress only weak antigens. Immunologists have long been interested inthe issue of what makes an antigen weak or strong. There have been twomajor hypotheses. Weak antigens may be poorly processed and fail to bepresented effectively to T cells. Alternatively, the number of T cellsin the organism with appropriate specificity might be inadequate for avigorous response (a so-called “hole in the repertoire”). Elucidation ofthe complex cellular process whereby antigenic peptides associate withMHC molecules for transport to the cell surface and presentation to Tcells has been one of the triumphs of modern immunology. Theseexperiments have clearly established that failure of presentation due toprocessing defects or competition from other peptides could render aparticular peptide less immunogenic. In contrast, it has, for technicalreasons, been more difficult to establish that the frequency of clonalrepresentation in the T cell repertoire is an important mechanism of lowresponsiveness. Recent studies demonstrating that the relationshipbetween immunodominant and cryptic peptides of a protein antigen changein T cell receptor transgenic mice suggest, however, that the relativefrequency of peptide-specific T cells can, indeed, be a determiningfactor in whether a particular peptide is cryptic or dominant in a Tcell response. This has encouraging implications for development ofvaccines. With present day methods, it would be a complex and difficultundertaking to modify the way in which antigenic peptides of a tumor areprocessed and presented to T cells. The relative frequency of a specificT cell population can, however, be directly and effectively increased byprior vaccination. This could, therefore, be the key manipulationrequired to render an otherwise cryptic response immunoprotective. Theseconsiderations of cryptic or sub-dominant antigens have specialrelevance in relation to possible immune evasion by tumors throughtolerance induction. Evidence has been presented to suggest thattumor-specific T cells in the tumor-bearing host are anergic, possiblyas a result of antigen presentation on non-professional APC (Morgan, D.J. et al., J. Immunol. 163:723-27 (1999); Sotomayor, E. M. et al., Proc.Natl. Acad. Sci. U.S.A. 96:11476-81 (1999); Lee, P. P. et al., NatureMedicine 5:677-85 (1999)). Prior tolerization of T cells specific forimmunodominant antigens of a tumor may, therefore, account for thedifficulty in developing successful strategies for immunotherapy ofcancer. These observations suggest that T cells specific forimmunodominant tumor antigens are less likely to be effective forimmunotherapy of established tumors because they are most likely to havebeen tolerized. It may, therefore, be that T cells specific forsub-dominant antigens or T cells that are initially present at a lowerfrequency would prove more effective because they have escaped thetolerizing influence of a growing tumor.

Another major concern for the development of broadly effective humanvaccines is the extreme polymorphism of HLA class I molecules. Class IMHC:cellular peptide complexes are the target antigens for specific CD8+CTLs. The cellular peptides, derived by degradation of endogenouslysynthesized proteins, are translocated into a pre-Golgi compartmentwhere they bind to class I MHC molecules for transport to the cellsurface. The CD8 molecule contributes to the avidity of the interactionbetween T cell and target by binding to the α3 domain of the class Iheavy chain. Since all endogenous proteins turn over, peptides derivedfrom any cytoplasmic or nuclear protein may bind to an MHC molecule andbe transported for presentation at the cell surface. This allows T cellsto survey a much larger representation of cellular proteins thanantibodies which are restricted to recognize conformational determinantsof only those proteins that are either secreted or integrated at thecell membrane.

The T cell receptor antigen binding site interacts with determinants ofboth the peptide and the surrounding MHC. T cell specificity must,therefore, be defined in terms of an MHC:peptide complex. Thespecificity of peptide binding to MHC molecules is very broad and ofrelatively low affinity in comparison to the antigen binding sites ofspecific antibodies. Class I-bound peptides are generally 8-10 residuesin length and accommodate amino acid side chains of restricted diversityat certain key positions that match pockets in the MHC peptide bindingsite. These key features of peptides that bind to a particular MHCmolecule constitute a peptide binding motif.

Hence, there exists a need for methods to facilitate the induction andisolation of T cells specific for human tumors, cancers and infectedcells and for methods to efficiently select the genes that encode themajor target antigens recognized by these T cells in the properMHC-context.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a novel polynucleotide, C35, that isdifferentially expressed in human breast and bladder carcinoma, and tothe encoded polypeptide of C35. Moreover, the present invention relatesto vectors, host cells, antibodies, and recombinant methods forproducing C35 polypeptides and polynucleotides. The present inventionfurther relates to the formulation and use of C35 polypeptides andpolynucleotides in immunogenic compositions to induce antibodies andcell-mediated immunity against target cells, such as tumor cells, thatexpress the C35 gene products. Also provided are diagnostic methods fordetecting disorders relating to the C35 genes and polypeptides,including use as a prognostic marker for carcinomas, such as humanbreast carcinoma, and therapeutic methods for treating such disorders.The invention further relates to screening methods for identifyingbinding partners of C35.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (Panels A-B). Panel A shows the DNA coding sequence (SEQ ID NO:1)of C35. The sequence immediately upstream of the predicted ATG startcodon is shown in lower case and conforms to the expected featuresdescribed by Kozak, M., J. Biol. Chem. 266(30):19867-19870 (1991). PanelB shows the deduced amino acid sequence (SEQ ID NO:2) of C35.

FIG. 2. (Panel A to C). Panel A: C35 is overexpressed in Breast tumorcell lines. Upper Panel: 300 ng of poly-A RNA from 3 week old humanthymus, normal breast epithelial cell line H16N2 from patient 21, and 4breast tumor cell lines derived one year apart from primary ormetastatic nodules of the same patient 21; 21NT, 21PT 21MT1, and 21MT2,was resolved on a 1% agarose/formaldehyde gel and transferred to aGeneScreen membrane. This blot was hybridized with a ³²P labeled C35probe. Hybridization was detected by exposing the blot to film for 15hours. Lower Panel: To quantitate RNA loading, the same blot wasstripped and re-hybridized with a ³²P labeled probe for Glyceraldehyde-3Phosphate Dehydrogenase (GAPDH). For each sample the C35 signal wasnormalized to the GAPDH signal. The numbers represent the foldexpression of C35 in each sample relative to H16N2. Panel B: C35 isexpressed at low levels in normal tissues. A Blot containing 1 microgramof poly-A RNA from each of the indicated adult normal tissues (Clontech)was hybridized with a ³²P labeled C35 probe. Hybridization was detectedby exposing the blot to film for 15 hours (upper panel), or 96 hours(lower panel). Panel C. C35 is overexpressed in primary Breast tumors. Ablot containing 2 micrograms of poly-A RNA from 3 primary infiltratingductal mammary carcinoma, T1, T2, T3 and 1 normal breast epithelium, N(Invitrogen) was hybridized with a ³²P labeled C35 probe. To normalizeloading a ³²P labeled beta-Actin probe was included in the hybridizationmix. Hybridization was detected by exposing the blot to film for 6hours. The numbers represent the fold expression of C35 in each samplerelative to normal breast epithelium.

FIG. 3. Expression of C35 in Breast Tumor Cell Lines. C35 isoverexpressed in different breast tumor cell lines. Upper Panel: 300 ngof poly-A RNA from BT474 (ATCC HYB-20, mammary ductal carcinoma), SKBR3(ATCC HTB-30, mammary adenocarcinoma), T47D (ATCC HTB-133, mammaryductal carcinoma), normal breast epithelial cell line H16N2 from patient21, and 21-NT breast tumor cell line derived from primary tumor noduleof the same patient 21 was resolved on a 1% agarose/formaldehyde gel andtransferred to a GeneScreen membrane. This blot was hybridized with a³²P labeled C35 probe. Hybridization was detected by exposing the blotto film for 15 hours. Lower Panel: To quantitate RNA loading, the sameblot was stripped and re-hybridized with a ³²P labeled probe forbeta-actin. For each sample the C35 signal was normalized to the actinsignal. The numbers represent the fold expression of C35 in each samplerelative to H16N2.

FIG. 4 (Panels A-C): Surface Expression of C35 Protein Detected by FlowCytometry. 1×10⁵ breast tumor cells were stained with 3.5 microliters ofantiserum raised in BALB/c mice against Line 1 mouse tumor cellstransduced with retrovirus encoding human C35 or control, pre-bleedBALB/c serum. After a 30 minute incubation, cells were washed twice withstaining buffer (PAB) and incubated with FITC-goat anti-mouse IgG (1μg/sample) for 30 minutes. Samples were washed and analyzed on an EPICSElite flow cytometer. Panel A: 21NT Panel B: SKBR3. Panel C: MDA-MB-231.These three breast tumor lines were selected to represent tumor cellsthat express high, intermediate and low levels of C35 RNA on Northernblots (see FIG. 3). Abbreviations: nms, ns; normal mouse serum; C35; C35immune serum.

FIG. 5 (Panels A and B). CML Selected Recombinant Vaccinia cDNA ClonesStimulate Tumor Specific CTL. Panel A: CML Selected vaccinia clones wereassayed for the ability, following infection of B/C.N, to stimulatetumor specific CTL to secrete interferon gamma. The amount of cytokinewas measured by ELISA, and is represented as OD490 (14). An OD490 of 1.4is approximately equal to 4 ng/ml of IFNg, and an OD490 of 0.65 isapproximately equal to 1 ng/ml of IFNg. Panel B: CML selected clonessensitize host cells to lysis by tumor specific CTL. Monolayers of B/C.Nin wells of a 6 well plate were infected with moi=1 of the indicatedvaccinia virus clones. After 14 hours of infection the infected cellswere harvested and along with the indicated control targets labeled with⁵¹Cr. Target cells were incubated with the indicated ratios of tumorspecific Cytotoxic T Lymphocytes for 4 hours at 37° C. and percentagespecific lysis was determined (15). This experiment was repeated atleast three times with similar results.

FIG. 6 (Panels A and B). The Tumor Antigen Is Encoded by a RibosomalProtein L3 Gene. Sequence of H2.16 and rpL3 from amino acid position 45to 56. Panel A: The amino acid (in single letter code) and nucleotidesequence of cDNA clone rpL3 (GenBank Accession no. Y00225). Panel B: Asingle nucleotide substitution at C170T of the H2.16 tumor cDNA is theonly sequence change relative to the published L3 ribosomal allele. Thissubstitution results in a T541 amino acid substitution in the protein.

FIG. 7 (Panels A and B). Identification of the Peptide EpitopeRecognized by the Tumor Specific CTL. Panel A: CML assay to identify thepeptide recognized by tumor specific CTL. Target cells were labeled with⁵¹Cr (15). During the ⁵¹Cr incubation samples of B/C.N cells wereincubated with 1 μM peptide L3₄₈₋₅₆(I54), 100 μM L3₄₈₋₅₆(T54) or 100 μMpeptide L3₄₅₋₅₄(I54). Target cells were incubated with the indicatedratios of tumor specific Cytotoxic T Lymphocytes for 4 hours at 37° C.and percentage specific lysis was determined. This experiment wasrepeated at least three times with similar results. Panel B: Titrationof peptide L3₄₈₋₅₆ (I54). Target cells were labeled with ⁵¹Cr. Duringthe ⁵¹Cr incubation samples of B/C.N cells were incubated either with nopeptide addition (D) or with the indicated concentrations (1 μM, 10 nM,1 nM) of L3₄₈₋₅₆(I54) (▪), BCA 39 cells were included as a positivecontrol (▴). Target cells were incubated with the indicated ratios ofTumor Specific Cytotoxic T Lymphocytes for 4 hours at 37° C. andpercentage specific lysis was determined. The experiment was repeatedtwice with similar results.

FIG. 8 (Panels A-C). Analysis of L3 Expressed by Each Cell Line. PanelA: Sau3AI map of published rpL3 and H2.16. Shown above is the Sau3AIrestriction map for the published ribosomal protein L3 gene (Top), andfor H2.16 (Bottom). Digestion of cDNA for the published L3 sequencegenerates fragments of 200, 355, 348, 289, and 84 bp. The pattern forH2.16 is identical except for an extra Sau3AI site at position 168caused by the C170T. This results in a 168 bp digestion product in placeof the 200 bp fragment. Panel B: The BCA tumors express both L3 alleles.RT-PCR products generated from each cell line or from vH2.16 weregenerated using L3 specific primers and then digested with Sau3AI, andresolved on a 3% agarose gel for 2 hours at 80 volts. Panel C: TheImmunogenic L3 allele is expressed at greatly reduced levels in B/C.N,BCB13, and Thymus. L3 specific RT-PCR products from each indicatedsample were generated using a ³²P end labeled 5 prime PCR primer. No PCRproduct was observed when RNA for each sample was used as template forPCR without cDNA synthesis, indicating that no sample was contaminatedwith genomic DNA. The PCR products were gel purified to ensure purity,digested with Sau3AI, and resolved on a 3% agarose gel for 15 hours at60 volts. No PCR product was observed in a control PCR sample that hadno template added to it. This result has been reproduced a total of 3times.

FIG. 9 (Panels A-C). Immunization with iL3 is Immunoprotective. Panel A:Immunization with H2.16 induces tumor specific CTL. Balb/c mice(2/group) were immunized by subcutaneous injection with 5×10⁶ pfu ofvH2.16, or control vector v7.5/tk. Seven days later splenocytes wereharvested and restimulated with peptide L348-56(I54) (26). Five daysfollowing the second restimulation the lymphocytes were tested in achromium release assay as described in FIG. 11. The L3₄₈₋₅₆(I54) peptidewas used at a 1 micromolar concentration, and the L3₄₈₋₅₆(T54) peptidewas used at a 100 micromolar concentration. Similar results wereobtained when the immunization experiment was repeated. Panels B and C:Female Balb/cByJ mice were immunized as indicated (27. The mice werechallenged by SC injection with 200,000 viable BCA 34 tumor cells intothe abdominal wall. Data is from day 35 post challenge. These data arerepresentative of 4 independent experiments.

FIG. 10 (Panels A and B). Panel A: C35 coding sequence with translation;5′ and 3′ untranslated regions are shown in lowercase letters. Thepredicted prenylation site, CVIL, at the 3′ terminus is boxed. Panel B:Genomic alignment of C35 gene on chromosome 17.

FIG. 11 (Panels A and B). C35 Expression in Breast Carcinoma. C35 waslabeled with ³²P in a random priming reaction and hybridized to Northernblots at 10⁶ cpm/ml. Each blot was stripped and re-probed with GAPDH orBeta-actin to normalize mRNA loads. The numbers indicate densitometryratios normalized against GAPDH/Beta-actin. A value of 1 has beenassigned to normal cell line H16N2, and all values are relative to thelevel of expression in the normal cell line. Panel A: C35 expression inbreast epithelial cell lines. Panel B: C35 expression in primary breasttissue/tumors. 300 ng mRNA was electrophoresed on 0.8% alkaline agarosegels, then blotted to Genescreen Plus, except leftmost panel of B loadedwith 1 μg mRNA from 3 primary tumors and 1 normal tissue control (RealTumor Blots, Invitrogen). Similar exposures are shown for all blots.

FIG. 12. C35 Expression in Bladder Carcinoma. C35 was labeled with ³²Pin a random priming reaction and hybridized to a Northern blot of tumorand normal RNA at 10⁶ cpm/ml. The blot was stripped and re-probed withBeta-actin to normalize mRNA loads. The numbers indicate densitometryratios normalized against Beta-actin. Values are relative to the levelof expression in the normal bladder samples. 300 ng mRNA waselectrophoresed on 0.8% alkaline agarose gels, then blotted toGenescreen Plus.

FIG. 13 (Panels A and B). FACS Analysis with Anti-C35 Antibodies. PanelA: Breast cell lines were stained with (top panel) sera from miceimmunized with Line 1 cells infected with C35 recombinant retrovirus,and (bottom panel) 2C3 purified monoclonal antibody or isotype control.Panel B: Bladder cell lines stained with 2C3 purified monoclonalantibody or isotype control.

FIG. 14. Inhibition of Tumor Growth in Presence of 2C3 Antibody. 21NTbreast tumor cells or H16N2 normal breast epithelial cells wereincubated with the indicated concentrations of 2C3 anti-C35 monoclonalantibody or a non-specific isotype control antibody. Cell growth wasmeasured by XTT assay following 72 hour incubation in the presence orabsence of antibodies.

FIG. 15 (Panels A and B). CTL stimulated with C35 expressing dendriticcells specifically lyse C35+ Breast (21NT) and Bladder (ppT11A3) tumorcell lines, with minimal activity against normal breast (MEC),immortalized non-tumorigenic breast (H16N2) and bladder (SV-HUC) celllines, or an NK sensitive cell line (K562). Panel A: T cell line 4 wasgenerated from normal human PBL. Panel B: T cell clone 10G3 was selectedfrom line 4 for C35-specific activity. Target cell lines MEC, ppT11A3and SV-HUC are naturally HLA-A2 positive. Target cell lines 21NT andH16N2 were transected with HLA-A2 to provide a required MHC restrictionelement.

FIG. 16 (Panels A and B). Cytokine Release from T Cell Clone 10G3 uponStimulation with Targets. Panel A: IFN-gamma secretion. Panel B:TNF-alpha secretion. Breast and bladder target cell lines weredistinguished by the presence or absence of expression of HLA-A2 and C35tumor antigen, an amino terminal 50 amino acid fragment of C35(C35-50aa), or the irrelevant mouse L3 ribosomal protein. Each markerwas either endogenously expressed or introduced by transfection of anHLA-A2.1 construct (pSV2.A2), or by infection with a vacciniarecombinant of C35 (vv.C35, vv.C35-50aa), L3 (vv.L3), or HLA-A2 (vv.A2)

FIG. 17 (Panels A (top) and B (Bottom)). Effect of anti-CD40 ligandantibody (anti-CD154) in blocking the reactivity of murine T cells tospecific transplantation antigens. Significant cytotoxicity was inducedagainst the control C3H alloantigens in both saline and anti-CD154treated mice (Panel A (top), whereas a cytotoxic response to C57B1/6 wasinduced in the saline treated mice but not the anti-CD154 treated mice(Panel B (bottom)).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding ofcertain terms used throughout this specification.

In the present invention, “isolated” refers to material removed from itsnative environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.

In the present invention, a “membrane” C35 protein is one expressed onthe cell surface through either direct or indirect association with thelipid bilayer, including, in particular, through prenylation of acarboxyl-terminal amino acid motif. Prenylation involves the covalentmodification of a protein by the addition of either a farnesyl orgeranylgeranyl isoprenoid. Prenylation occurs on a cysteine residuelocated near the carboxyl-terminus of a protein. The C35 polypeptidecontains the amino acids Cys-Val-Ile-Leu at positions 112-115, with theLeu being the C terminal residue of the polypeptide. The motifCys-X-X-Leu, where “X” represents any aliphatic amino acid, results inthe addition of a 20 carbon geranylgeranyl group onto the Cys residue.Generally, following addition of this lipid the three terminal aminoacid residues are cleaved off the polypeptide, and the lipid group ismethylated. Prenylation promotes the membrane localization of mostproteins, with sequence motifs in the polypeptide being involved indirecting the prenylated protein to the plasma, nuclear, or golgimembranes. Prenylation plays a role in protein-protein interactions, andmany prenylated proteins are involved in signal transduction. Examplesof prenylated proteins include Ras and the nuclear lamin B. (Zhang, F.L. and Casey, P. J., Ann. Rev. Biochem. 65:241-269 (1996)). The C35protein has been detected on the surface of two breast tumor cell linesby fluorescence analysis employing as a primary reagent a mouseanti-human C35 antiserum (FIG. 4).

In the present invention, a “secreted” C35 protein refers to a proteincapable of being directed to the ER, secretory vesicles, or theextracellular space as a result of a signal sequence, as well as a C35protein released into the extracellular space without necessarilycontaining a signal sequence. If the C35 secreted protein is releasedinto the extracellular space, the C35 secreted protein can undergoextracellular processing to produce a “mature” C35 protein. Release intothe extracellular space can occur by many mechanisms, includingexocytosis and proteolytic cleavage.

As used herein, a C35 “polynucleotide” refers to a molecule having anucleic acid sequence contained in SEQ ID NO: 1. For example, the C35polynucleotide can contain the nucleotide sequence of the full lengthcDNA sequence, including the 5′ and 3′ untranslated sequences, thecoding region, with or without the signal sequence, the secreted proteincoding region, as well as fragments, epitopes, domains, and variants ofthe nucleic acid sequence. Moreover, as used herein, a C35 “polypeptide”refers to a molecule having the translated amino acid sequence generatedfrom the polynucleotide as broadly defined.

In specific embodiments, the polynucleotides of the invention are lessthan 300 nt, 200 nt, 100 nt, 50 nt, 15 nt, 10 nt, or 7 nt in length. Ina further embodiment, polynucleotides of the invention comprise at least15 contiguous nucleotides of C35 coding sequence, but do not compriseall or a portion of any C35 intron. In another embodiment, the nucleicacid comprising C35 coding sequence does not contain coding sequences ofa genomic flanking gene (i.e., 5′ or 3′ to the C35 gene in the genome).

In the present invention, the full length C35 coding sequence isidentified as SEQ ID NO: 1.

A C35 “polynucleotide” also refers to isolated polynucleotides whichencode the C35 polypeptides, and polynucleotides closely relatedthereto.

A C35 “polynucleotide” also refers to isolated polynucleotides whichencode the amino acid sequence shown in SEQ ID NO: 2, or a biologicallyactive fragment thereof.

A C35 “polynucleotide” also includes those polynucleotides capable ofhybridizing, under stringent hybridization conditions, to sequencescontained in SEQ ID NO: 1, the complement thereof, or the cDNA withinthe deposited clone. “Stringent hybridization conditions” refers to anovernight incubation at 42Λ in a solution comprising 50% formamide,5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1×SSC at about 65Λ

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA), or to a complementarystretch of T (or U) residues, would not be included in the definition of“polynucleotide,” since such a polynucleotide would hybridize to anynucleic acid molecule containing a poly (A) stretch or the complementthereof (e.g., practically any double-stranded cDNA clone).

The C35 polynucleotide can be composed of any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. For example, C35 polynucleotides can be composed of single-and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the C35 polynucleotides can be composed of triple-strandedregions comprising RNA or DNA or both RNA and DNA. C35 polynucleotidesmay also contain one or more modified bases or DNA or RNA backbonesmodified for stability or for other reasons. “Modified” bases include,for example, tritylated bases and unusual bases such as inosine. Avariety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

C35 polypeptides can be composed of amino acids joined to each other bypeptide bonds or modified peptide bonds, i.e., peptide isosteres, andmay contain amino acids other than the 20 gene-encoded amino acids. TheC35 polypeptides may be modified by either natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theC35 polypeptide, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini. It will be appreciatedthat the same type of modification may be present in the same or varyingdegrees at several sites in a given C35 polypeptide. Also, a given C35polypeptide may contain many types of modifications. C35 polypeptidesmay be branched, for example, as a result of ubiquitination, and theymay be cyclic, with or without branching. Cyclic, branched, and branchedcyclic C35 polypeptides may result from posttranslation naturalprocesses or may be made by synthetic methods. Modifications includeacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. (See, for instance, Proteins—StructureAnd Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993); Posttranslational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12(1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al.,Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO: 1” refers to a C35 polynucleotide sequence while “SEQ ID NO:2” refers to a C35 polypeptide sequence.

A C35 polypeptide “having biological activity” refers to polypeptidesexhibiting activity similar to, but not necessarily identical to, anactivity of a C35 polypeptide, including mature forms, as measured in aparticular biological assay, with or without dose dependency. In thecase where dose dependency does exist, it need not be identical to thatof the C35 polypeptide, but rather substantially similar to thedose-dependence in a given activity as compared to the C35 polypeptide(i.e., the candidate polypeptide will exhibit greater activity or notmore than about 25-fold less and, preferably, not more than abouttenfold less activity, and most preferably, not more than aboutthree-fold less activity relative to the C35 polypeptide.)

C35 Polynucleotides and Polypeptides

A 348 base pair fragment of C35 was initially isolated by subtractivehybridization of poly-A RNA from tumor and normal mammary epithelialcell lines derived from the same patient with primary and infiltratingintraductal mammary carcinoma. Band, V. et al., Cancer Res. 50:7351-7357(1990). Employing primers based on this sequence and that of anoverlapping EST sequence (Accession No. W57569), a cDNA that includesthe full-length C35 coding sequence was then amplified and cloned fromthe BT-20 breast tumor cell line (ATCC, HTB-19). This C35 cDNA containsthe entire coding region identified as SEQ ID NO:1. The C35 cloneincludes, in addition to the 348 bp coding sequence, 167 bp of 3′untranslated region. The open reading frame begins at an N-terminalmethionine located at nucleotide position 1, and ends at a stop codon atnucleotide position 348 (FIG. 1). A representative clone containing allor most of the sequence for SEQ ID NO:1 was deposited with the AmericanType Culture Collection (“ATCC”) on Aug. 1, 2000, and was given the ATCCDeposit Number PTA-2310. The ATCC is located at 10801 UniversityBoulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was madepursuant to the terms of the Budapest Treaty on the internationalrecognition of the deposit of microorganisms for purposes of patentprocedure.

Therefore, SEQ ID NO: 1 and the translated SEQ ID NO: 2 are sufficientlyaccurate and otherwise suitable for a variety of uses well known in theart and described further below. For instance, SEQ ID NO: 1 is usefulfor designing nucleic acid hybridization probes that will detect nucleicacid sequences contained in SEQ ID NO: 1 or the cDNA contained in thedeposited clone. These probes will also hybridize to nucleic acidmolecules in biological samples, thereby enabling a variety of forensicand diagnostic methods of the invention. Similarly, polypeptidesidentified from SEQ ID NO:2 may be used to generate antibodies whichbind specifically to C35, or to stimulate T cells which are specific forC35 derived peptides in association with MHC molecules on the cellsurface.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:1 and the predicted translated amino acid sequence identified as SEQID NO:2. The nucleotide sequence of the deposited C35 clone can readilybe determined by sequencing the deposited clone in accordance with knownmethods. The predicted C35 amino acid sequence can then be verified fromsuch deposits. Moreover, the amino acid sequence of the protein encodedby the deposited clone can also be directly determined by peptidesequencing or by expressing the protein in a suitable host cellcontaining the deposited human C35 cDNA, collecting the protein, anddetermining its sequence.

The present invention also relates to the C35 gene corresponding to SEQID NO:1, or the deposited clone. The C35 gene can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include preparing probes or primers from thedisclosed sequence and identifying or amplifying the C35 gene fromappropriate sources of genomic material.

Also provided in the present invention are species homologs of C35.Species homologs may be isolated and identified by making suitableprobes or primers from the sequences provided herein and screening asuitable nucleic acid source for the desired homologue.

By “C35 polypeptide(s)” is meant all forms of C35 proteins andpolypeptides described herein. The C35 polypeptides can be prepared inany suitable manner. Such polypeptides include isolated naturallyoccurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. Means for preparing such polypeptides arewell understood in the art.

The C35 polypeptides may be in the form of the membrane protein or asecreted protein, including the mature form, or may be a part of alarger protein, such as a fusion protein (see below). It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification, such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

C35 polypeptides are preferably provided in an isolated form, andpreferably are substantially purified. A recombinantly produced versionof a C35 polypeptide, including the secreted polypeptide, can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). C35 polypeptides also can be purifiedfrom natural or recombinant sources using antibodies of the inventionraised against the C35 protein in methods which are well known in theart.

Polynucleotide and Polypeptide Variants

“Variant” refers to a polynucleotide or polypeptide differing from theC35 polynucleotide or polypeptide, but retaining essential propertiesthereof. Generally, variants are overall closely similar, and, in manyregions, identical to the C35 polynucleotide or polypeptide.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the C35polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence shown of SEQ ID NO:1, the ORF (open reading frame),or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to anucleotide sequence of the presence invention can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al., Comp. App. Biosci.6:237-245 (1990). In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byconverting U's to T's. The result of said global sequence alignment isin percent identity. Preferred parameters used in a FASTDB alignment ofDNA sequences to calculate percent identity are: Matrix=Unitary,k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization GroupLength=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, WindowSize=500 or the length of the subject nucleotide sequence, whichever isshorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2 or to the amino acid sequence encodedby deposited DNA clone, can be determined conventionally using knowncomputer programs. A preferred method for determining the best overallmatch between a query sequence (a sequence of the present invention) anda subject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al., Comp. App. Biosci. 6:237-245 (1990). In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to be made forthe purposes of the present invention.

The C35 variants may contain alterations in the coding regions,non-coding regions, or both. Especially preferred are polynucleotidevariants containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Nucleotide variants produced by silentsubstitutions due to the degeneracy of the genetic code are preferred.Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.C35 polynucleotide variants can be produced for a variety of reasons,e.g., to optimize codon expression for a particular host (change codonsin the human mRNA to those preferred by a bacterial host such as E.coli).

Naturally occurring C35 variants are called “allelic variants,” andrefer to one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985).) Also, allelic variants can occur as“tandem alleles” which are highly homologous sequences that occur atdifferent loci on chromosomes of an organism. These allelic variants canvary at either the polynucleotide and/or polypeptide level.Alternatively, non-naturally occurring variants may be produced bymutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the C35 polypeptides. For instance, one or more aminoacids can be deleted from the N-terminus or C-terminus of the secretedprotein without substantial loss of biological function. The authors ofRon et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGFproteins having heparin binding activity even after deleting 3, 8, or 27amino-terminal amino acid residues. Similarly, Interferon gammaexhibited up to ten times higher activity after deleting 8-10 amino acidresidues from the carboxy terminus of this protein (Dobeli et al., J.Biotechnology 7:199-216 (1988)).

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” (See, Abstract.) In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Thus, the invention further includes C35 polypeptide variants which showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as to have little effect on activity.For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., Science247:1306-1310 (1990), wherein the authors indicate that there are twomain strategies for studying the tolerance of an amino acid sequence tochange.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and H is; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

Besides conservative amino acid substitution, variants of C35 include(i) substitutions with one or more of the non-conserved amino acidresidues, where the substituted amino acid residues may or may not beone encoded by the genetic code, or (ii) substitution with one or moreof amino acid residues having a substituent group, or (iii) fusion ofthe mature polypeptide with another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), or (iv) fusion of the polypeptide withadditional amino acids, such as an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

For example, C35 polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Polynucleotide and Polypeptide Fragments

In the present invention, a “polynucleotide fragment” refers to a shortpolynucleotide having a nucleic acid sequence contained in the depositedclone or shown in SEQ ID NO:1. The short nucleotide fragments arepreferably at least about 15 nt, and more preferably at least about 20nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length. A fragment “at least 20 ntin length,” for example, is intended to include 20 or more contiguousbases from the cDNA sequence contained in the deposited clone or thenucleotide sequence shown in SEQ ID NO:1. These nucleotide fragments areuseful as diagnostic probes and primers as discussed herein. Of course,larger fragments (e.g., at least 50, 100, 150, 200, 250, 300nucleotides) are preferred.

Moreover, representative examples of C35 polynucleotide fragmentsinclude, for example, fragments having a sequence from about nucleotidenumber 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, or 301 to theend of SEQ ID NO:1 or the cDNA contained in the deposited clone. In thiscontext “about” includes the particularly recited ranges, larger orsmaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus orat both termini. Preferably, these fragments encode a polypeptide whichhas biological activity. More preferably, these polynucleotides can beused as probes or primers as discussed herein.

In the present invention, a “polypeptide fragment” refers to a shortamino acid sequence contained in SEQ ID NO:2 or encoded by the cDNAcontained in the deposited clone. Protein fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, or 101 to the end of the codingregion. Moreover, polypeptide fragments can comprise 9, 15, 20, 30, 40,50, 60, 70, 80, 90, or 100 amino acids in length. In this context“about” includes the particularly recited ranges, larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes.

Preferred polypeptide fragments include the secreted C35 protein as wellas the mature form. Further preferred polypeptide fragments include thesecreted C35 protein or the mature form having a continuous series ofdeleted residues from the amino or the carboxy terminus, or both.

As mentioned above, even if deletion of one or more amino acids from theN-terminus of a protein results in modification or loss of one or morebiological functions of the protein, other biological activities maystill be retained. Thus, the ability of shortened C35 muteins to induceand/or bind to antibodies which recognize the complete or mature formsof the polypeptides generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the N-terminus. Whether a particular polypeptide lackingN-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a C35 muteinwith a large number of deleted N-terminal amino acid residues may retainsome biological or immunogenic activities. In fact, peptides composed ofas few as 9 C35 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the C35 aminoacid sequence shown in SEQ ID NO:2, up to the Threonine residue atposition number 105 and polynucleotides encoding such polypeptides.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other biological activitiesmay still be retained. Thus, the ability of the shortened C35 mutein toinduce and/or bind to antibodies which recognize the complete or matureforms of the polypeptide generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the C-terminus. Whether a particular polypeptide lackingC-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a C35 muteinwith a large number of deleted C-terminal amino acid residues may retainsome biological or immunogenic activities.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the C35 polypeptide shown in SEQ ID NO:2, up to the valineresidue at position number 10, and polynucleotides encoding suchpolypeptides

Moreover, the invention also provides polypeptides having one or moreamino acids deleted from both the amino and the carboxyl termini. Inpreferred embodiments, the invention is directed to polypeptides havingresidues: S-9 to V-17; V-10 to V-17; E-16 to V-23; E-16 to R-24; E-16 toI-25; S-21 to F-35; C-30 to T-38; E-31 to Y-39; E-36 to A-43; A-37 toA-45; A-37 to V-46; Y-39 to V-46; S-44 to I-53; A-45 to I-53; G-52 toL-59; E-54 to T-62; S-57 to F-75; R-58 to I-67; G-61 to I-69; G-63 toF-83; E-66 to L-73; E-66 to V-74; F-83 to E-103; D-88 to A-96; L-89 toA-96; A-92 to T-101; R-95 to L-102; A-96 to K-104; K-104 to V-113; I-105to V-113; I-105 to I-114 of SEQ ID NO:2, and polynucleotides encodingsuch polypeptides.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases.

The human EST sequences referred to below were identified in a BLASTsearch of the EST database. These sequences are believed to be partialsequences of the cDNA inserts identified in the recited GenBankaccession numbers. No homologous sequences were identified in a searchof the annotated GenBank database. The Expect value (E) is a parameterthat describes the number of hits one can “expect” to see just by chancewhen searching a database of a particular size. It decreasesexponentially with the Score (S) that is assigned to a match between twosequences. Essentially, the E value describes the random backgroundnoise that exists for matches between sequences. In BLAST 2.0, theExpect value is also used instead of the P value (probability) to reportthe significance of matches. For example, an E value of 1 assigned to ahit can be interpreted as meaning that in a database of the current sizeone might expect to see 1 match with a similar score simply by chance.

For example, the following sequences are related to SEQ ID NO:1, GenBankAccession Nos.: AA971857 (SEQ ID NO:3); W57569 (SEQ ID NO:4); AI288765(SEQ ID NO:5); W65390 (SEQ ID NO:6); W37432 (SEQ ID NO: 7); N42748 (SEQID NO:8); AA971638 (SEQ ID NO:9); R22331 (SEQ ID NO:10); AA308370 (SEQID NO:11); AA285089 (SEQ ID NO:12); R68901 (SEQ ID NO:13); AA037285 (SEQID NO:14); H94832 (SEQ ID NO:15); H96058 (SEQ ID NO:16); H56522 (SEQ IDNO:17); AA935328 (SEQ ID NO: 18); AW327450 (SEQ ID NO:19); AW406075 (SEQID NO:20); AW406223 (SEQ ID NO:21); AI909652 (SEQ ID NO:22); AA026773(SEQ ID NO: 23); H96055 (SEQ ID NO:24); H12836 (SEQ ID NO:25); R22401(SEQ ID NO:26); N34596 (SEQ ID NO:27); W32121 (SEQ ID NO:28); T84927(SEQ ID NO:29); R63575 (SEQ ID NO:30); R23139 (SEQ ID NO:31); AA337071(SEQ ID NO:32); AA813244 (SEQ ID NO:33); AA313422 (SEQ ID NO:34); N31910(SEQ ID NO:35); N42693 (SEQ ID NO:36); N32532 (SEQ ID NO:37); AA375119(SEQ ID NO:38); R32153 (SEQ ID NO:39); R23369 (SEQ ID NO:40); AA393628(SEQ ID NO:41); H12779 (SEQ ID NO:42); AI083674 (SEQ ID NO:43); AA284919(SEQ ID NO:44); AA375286 (SEQ ID NO:45); AA830592 (SEQ ID NO:46); H95363(SEQ ID NO:47); T92052 (SEQ ID NO:48); AI336555 (SEQ ID NO:49); AI285284(SEQ ID NO:50); AA568537 (SEQ ID NO:51); AI041967 (SEQ ID NO:52); W44577(SEQ ID NO:53); R22332 (SEQ ID NO:54); N27088 (SEQ ID NO:55); H96418(SEQ ID NO:56); AI025384 (SEQ ID NO:57); AA707623 (SEQ ID NO:58);AI051009 (SEQ ID NO:59); AA026774 (SEQ ID NO:60); W51792 (SEQ ID NO:61);AI362693 (SEQ ID NO:62); AA911823 (SEQ ID NO:63); H96422 (SEQ ID NO:64);AI800991 (SEQ ID NO:65); AI525314 (SEQ ID NO:66); AI934846 (SEQ IDNO:67); AI937133 (SEQ ID NO:68); AWO06797 (SEQ ID NO:69); AI914716 (SEQID NO:70); AI672936 (SEQ ID NO:71); W61294 (SEQ ID NO:72); AI199227 (SEQID NO:73); AI499727 (SEQ ID NO:74); R32154 (SEQ ID NO:75); AI439771 (SEQID NO:76); AA872671 (SEQ ID NO:77); AA502178 (SEQ ID NO:78); N26715 (SEQID NO:79); AA704668 (SEQ ID NO:80); R68799 (SEQ ID NO:81); H56704 (SEQID NO:82); AI360416 (SEQ ID NO:83).

Thus, in one embodiment the present invention is directed topolynucleotides comprising the polynucleotide fragments and full-lengthpolynucleotide (e.g. the coding region) described herein exclusive ofone or more of the above-recited ESTs.

Also preferred are C35 polypeptide and polynucleotide fragmentscharacterized by structural or functional domains. Preferred embodimentsof the invention include fragments that comprise MHC binding epitopesand prenylation sites.

Other preferred fragments are biologically active C35 fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the C35 polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

Epitopes & Antibodies

Cellular peptides derived by degradation of endogenously synthesizedproteins are translocated into a pre-Golgi compartment where they bindto Class I MHC molecules for transport to the cell surface. These classI MHC:peptide complexes are the target antigens for specific CD8+cytotoxic T cells. Since all endogenous proteins “turn over,” peptidesderived from any cytoplasmic or nuclear protein may bind to an MHCmolecule and be transported for presentation at the cell surface. Thisallows T cells to survey a much larger representation of cellularproteins than antibodies which are restricted to recognizeconformational determinants of only those proteins that are eithersecreted or integrated at the cell membrane. The T cell receptor antigenbinding site interacts with determinants of both the peptide and thesurrounding MHC. T cell specificity must, therefore, be defined in termsof an MHC:peptide complex. The specificity of peptide binding to MHCmolecules is very broad and of relatively low affinity in comparison tothe antigen binding site of specific antibodies. Class I-bound peptidesare generally 8-10 residues in length that accommodate amino acid sidechains of restricted diversity at certain key positions that matchpockets in the MHC peptide binding site. These key features of peptidesthat bind to a particular MHC molecule constitute a peptide bindingmotif.

A number of computer algorithms have been described for identificationof peptides in a larger protein that may satisfy the requirements ofpeptide binding motifs for specific MHC class I or MHC class IImolecules. Because of the extensive polymorphism of MHC molecules,different peptides will often bind to different MHC molecules. Tables1-3 list C35 peptides predicted to be MHC binding peptides using threedifferent algorithms. Specifically, Tables 1 and 5 list C35 HLA Class Iand II epitopes predicted using the rules found at the SYFPEITHI websiteand are based on the book “MHC Ligands and Peptide Motifs” by Rammensee,H. G., Bachmann, J. and Stevanovic, S. (Chapman & Hall, New York 1997).Table 2 lists predicted MHC binding peptides derived from the C35sequence using the NIH BIMAS program available on the web. Finally,Tables 3 and 6 list predicted C35 peptides identified by the Tepitopeprogram, a program for prediction of peptides that may bind to multipledifferent MHC class II molecules. Using Tepitope, four C35 peptides wereidentified as likely candidates for binding to a variety of HLA class IImolecules. These peptides are, in general, longer than those binding toHLA class I and more degenerate in terms of binding to multiple HLAclass II molecules. Unless expressly noted otherwise, all peptidesequences listed in Tables 1-6 refer to C35 peptide sequences appearingin SEQ ID NO:2 at the amino acid positions noted.

TABLE 1 C35 peptides predicted by SYFPEITHI website (score reflectsligation strength): Class I MHC HLA-A*0201 nonamers Position 1 2 3 4 5 67 8 9 Score   9 S V A P P P E E V 23  88 D L I E A I R R A 21  37 A T YL E L A S A 19  97 S N G E T L E K I 18 105 I T N S R P P C V 18   2 S GE P G Q T S V 17  45 A V K E Q Y P G I 17  38 T Y L E L A S A V 16  61 GT G A F E I E I 16  85 Y E K D L I E A I 16  65 F E I E I N G Q L 15 107N S R P P C V I L 15  41 E L A S A V K E Q 14  58 R L G G T G A F E 14 59 L G G T G A F E I 14  66 E I E I N G Q L V 14  68 E I N G Q L V F S14  81 G G F P Y E K D L 14  94 R R A S N G E T L 14 HLA-A*0201 decamersPosition 1 2 3 4 5 6 7 8 9 Score  58 R L G G T G A F E I 22  96 A S N GE T L E K I 19 104 K I T N S R P P C V 19  37 A T Y L E L A S A V 18  17V E P G S G V R I V 17  33 C G F E A T Y L E L 16  44 S A V K E Q Y P GI 16  92 A I R R A S N G E T 16  39 Y L E L A S A V K E 15  53 I E I E SR L G G T 15  65 F E I E I N G Q L V 15 105 I T N S R P P C V I 15   1 MS G E P G Q T S V 14  63 G A F E I E I N G Q 14  68 E I N G Q L V F S K14  69 I N G Q L V F S K L 14  83 F P Y E K D L I E A 14  88 D L I E A IR R A S 14  93 I R R A S N G E T L 14  72 Q L V F S K L E N G 13  89 L IE A I R R A S N 13   8 T S V A P P P E E V 12  16 E V E P G S G V R I 12 50 Y P G I E I E S R L 12  60 G G T G A F E I E I 12  81 G G F P Y E KD L I 12 106 T N S R P P C V I L 12 HLA-A*0203 nonamers Position 1 2 3 45 6 7 8 9 Score  35 F E A T Y L E L A 12 HLA-A*0203 decamers Position 12 3 4 5 6 7 8 9 Score  36 E A T Y L E L A S A 18 HLA-A1 nonamersPosition 1 2 3 4 5 6 7 8 9 Score  77 K L E N G G F P Y 29   2 S G E P GQ T S V 18  21 S G V R I V V E Y 18  16 E V E P G S G V R 17  29 Y C E PC G F E A 17  42 L A S A V K E Q Y 17  31 E P C G F E A T Y 16  34 G F EA T Y L E L 16  39 Y L E L A S A V K 14  84 P Y E K D L I E A 14  66 E IE I N G Q L V 13  13 P P E E V E P G S 12  46 V K E Q Y P G I E 12  52 GI E I E S R L G 12  96 A S N G E T L E K 12 HLA-A1 decamers Position 1 23 4 5 6 7 8 9 Score  20 G S G V R I V V E Y 20  29 Y C E P C G F E A T19  76 S K L E N G G F P Y 18   2 S G E P G Q T S V A 17  52 G I E I E SR L G G 17  66 E I E I N G Q L V F 17  41 E L A S A V K E Q Y 16  46 V KE Q Y P G I E I 16  16 E V E P G S G V R I 15  30 C E P C G F E A T Y 15 39 Y L E L A S A V K E 15  77 K L E N G G F P Y E 14  86 E K D L I E AI R R 14  98 N G E T L E K I T N 14  34 G F E A T Y L E L A 12  64 A F EI E I N G Q L 12 101 T L E K I T N S R P 12 HLA-A26 nonamers Position 12 3 4 5 6 7 8 9 Score  68 E I N G Q L V F S 24 100 E T L E K I T N S 24 88 D L I E A I R R A 23  54 E I E S R L G G T 22  41 E L A S A V K E Q21  45 A V K E Q Y P G I 20  31 E P C G F E A T Y 19  34 G F E A T Y L EL 19  73 L V F S K L E N G 19  16 E V E P G S G V R 18  77 K L E N G G FP Y 18  66 E I E I N G Q L V 17  21 S G V R I V V E Y 16  37 A T Y L E LA S A 16  24 R I V V E Y C E P 15   9 S V A P P P E E V 14  22 G V R I VV E Y C 14  51 P G I E I E S R L 14  70 N G Q L V F S K L 14  57 S R L GG T G A F 13  65 F E I E I N G Q L 13  25 I V V E Y C E P C 12  48 E Q YP G I E I E 12  67 I E I N G Q L V F 12  75 F S K L E N G G F 12  81 G GF P Y E K D L 12 104 K I T N S R P P C 12 105 I T N S R P P C V 12HLA-A26 decamers Position 1 2 3 4 5 6 7 8 9 Score  41 E L A S A V K E QY 27  66 E I E I N G Q L V F 26  68 E I N G Q L V F S K 23  26 V V E Y CE P C G F 21  16 E V E P G S G V R I 20  88 D L I E A I R R A S 19 100 ET L E K I T N S R 19  74 V F S K L E N G G F 18  33 C G F E A T Y L E L17  54 E I E S R L G G T G 17  56 E S R L G G T G A F 17  20 G S G V R IV V E Y 16  31 E P C G F E A T Y L 16  64 A F E I E I N G Q L 15  69 I NG Q L V F S K L 15  61 G T G A F E I E I N 14  73 L V F S K L E N G G 14  9 S V A P P P E E V E 13  25 I V V E Y C E P C G 13  45 A V K E Q Y PG I E 13  72 Q L V F S K L E N G 13  77 K L E N G G F P Y E 13  79 E N GG F P Y E K D 13   4 E P G Q T S V A P P 12   7 Q T S V A P P P E E 12 30 C E P C G F E A T Y 12  36 E A T Y L E L A S A 12  37 A T Y L E L AS A V 12  76 S K L E N G G F P Y 12  89 L I E A I R R A S N 12 HLA-A3nonamers Position 1 2 3 4 5 6 7 8 9 Score  39 Y L E L A S A V K 28  77 KL E N G G F P Y 25  16 E V E P G S G V R 24  58 R L G G T G A F E 22  67I E I N G Q L V F 19  96 A S N G E T L E K 18  92 A I R R A S N G E 17  9 S V A P P P E E V 16 101 T L E K I T N S R 16  22 G V R I V V E Y C15  31 E P C G F E A T Y 15  45 A V K E Q Y E G I 15  72 Q L V F S K L EN 15  21 S G V R I V V E Y 14  68 E I N G Q L V F S 14  69 I N G Q L V FS K 14  88 D L I E A I R R A 14  91 E A I R R A S N G 14  25 I V V E Y CE P C 13  37 A T Y L E L A S A 13  55 I E S R L G G T G 13  57 S R L G GT G A F 13  79 E N G G F P Y E K 13  87 K D L I E A I R R 13 104 K I T NS R P P C 13  24 R I V V E Y C E P 12  42 L A S A V K E Q Y 12  66 E I EI N G Q L V 12  89 L I E A I R R A S 12  90 I E A I R R A S N 12  94 R RA S N G E T L 12 HLA-A3 decamers Position 1 2 3 4 5 6 7 8 9 Score  68 EI N G Q L V F S K 22  16 E V E P G S G V R I 20  38 T Y L E L A S A V K20  41 E L A S A V K E Q Y 20  66 E I E I N G Q L V F 20   9 S V A P P PE E V E 19  58 R L G G T G A F E I 19  39 Y L E L A S A V K E 18  92 A IR R A S N G E T 18  95 R A S N G E T L E K 18  45 A V K E Q Y P G I E 17 54 E I E S R L G G T G 16  88 D L I E A I R R A S 16  89 L I E A I R RA S N 16  26 V V E Y C E P C G F 15  37 A T Y L E L A S A V 15  22 G V RI V V E Y C E 14  77 K L E N G G F P Y E 14  93 I R R A S N G E T L 14 25 I V V E Y C E P C G 13  30 C E P C G F E A T Y 13  52 G I E I E S RL G G 13  76 S K L E N G G F P Y 13  78 L E N G G F P Y E K 13 101 T L EK I T N S R P 13 104 K I T N S R P P C V 13  24 R I V V E Y C E P C 12 72 Q L V F S K L E N G 12 HLA-B*0702 nonamers Position 1 2 3 4 5 6 7 89 Score  18 E P G S G V R I V 19 107 N S R P P C V I L 18   4 E P G Q TS V A P 15  11 A P P P E E V E P 15  31 E P C G F E A T Y 14  34 G F E AT Y L E L 13  94 R R A S N G E T L 13  12 P P P E E V E P G 12  19 P G SG V R I V V 12  32 P C G F E A T Y L 12  83 F P Y E K D L I E 12 106 T NS R P P C V I 12 HLA-B*0702 decamers Position 1 2 3 4 5 6 7 8 9 Score 31 E P C G F E A T Y L 24  50 Y P G I E I E S R L 21  18 E P G S G V RI V V 20  83 F P Y E K D L I E A 16   4 E P G Q T S V A P P 15  11 A P PP E E V E P G 15  93 I R R A S N G E T L 14 106 T N S R P P C V I L 14 69 I N G Q L V F S K L 13  33 C G F E A T Y L E L 12  64 A F E I E I NG Q L 12 HLA-B*08 octamers Position 1 2 3 4 5 6 7 8 9 Score  83 F P Y EK D L I 25  66 E I E I N G Q L 16  52 G I E I E S R L 15  18 E P G S G VR I 14  54 E I E S R L G G 14  91 E A I R R A S N 14  95 R A S N G E T L14 100 E T L E K I T N 14  33 C G F E A T Y L 12  45 A V K E Q Y P G 12 58 R L G G T G A F 12  68 E I N G Q L V F 12  71 G Q L V F S K L 12  75F S K L E N G G 12  82 G F P Y E K D L 12 107 N S R P P C V I 12 108 S RP P C V I L 12 HLA-B*08 nonamers Position 1 2 3 4 5 6 7 8 9 Score  75 FS K L E N G G F 19  83 F P Y E K D L I E 19  45 A V K E Q Y P G I 18  85Y E K D L I E A I 18 107 N S R P P C V I L 17 100 E T L E K I T N S 15 54 E I E S R L G G T 14  65 F E I E I N G Q L 14  91 E A I R R A S N G14  20 G S G V R I V V E 12  34 G F E A T Y L E L 12  51 P G I E I E S RL 12  81 G G F P Y E K D L 12 HLA-B*1510 nonamers Position 1 2 3 4 5 6 78 9 Score 107 N S R P P C V I L 15  34 G F E A T Y L E L 13  51 P G I EI E S R L 13  81 G G F P Y E K D L 13  94 R R A S N G E T L 13HLA-B*2705 nonamers Position 1 2 3 4 5 6 7 8 9 Score  57 S R L G G T G AF 26  94 R R A S N G E T L 25  67 I E I N G Q L V F 19  87 K D L I E A IR R 19  51 P G I E I E S R L 17  81 G G F P Y E K D L 17  65 F E I E I NG Q L 16  69 I N G Q L V F S K 16  96 A S N G E T L E K 16  16 E V E P GS G V R 15  34 G F E A T Y L E L 15  50 Y P G I E I E S R 15  70 N G Q LV F S K L 15 101 T L E K I T N S R 15  23 V R I V V E Y C E 14  32 P C GF E A T Y L 14  39 Y L E L A S A V K 14  79 E N G G F P Y E K 14  93 I RR A S N G E T 14  21 S G V R I V V E Y 13  27 V E Y C E P C G F 13  75 FS K L E N G G F 13  86 E K D L I E A I R 13 107 N S R P P C V I L 13  17V E P G S G V R I 12  31 E P C G F E A T Y 12  77 K L E N G G F P Y 12HLA-B*2709 nonamers Position 1 2 3 4 5 6 7 8 9 Score  94 R R A S N G E TL 25  57 S R L G G T G A F 20  81 G G F P Y E K D L 16  34 G F E A T Y LE L 14  51 P G I E I E S R L 13  65 F E I E I N G Q L 13  23 V R I V V EY C E 12 107 N S R P P C V I L 12 HLA-B*5101 nonamers Position 1 2 3 4 56 7 8 9 Score  18 E P G S G V R I V 21  81 G G F P Y E K D L 21  51 P GI E I E S R L 20  70 N G Q L V F S K L 20  19 P G S G V R I V V 19  31 EP C G F E A T Y 19   2 S G E P G Q T S V 18  42 L A S A V K E Q Y 18  59L G G T G A F E I 18  21 S G V R I V V E Y 14  83 F P Y E K D L I E 14 97 S N G E T L E K I 14  13 P P E E V E P G S 13  38 T Y L E L A S A V13  45 A V K E Q Y P G I 13  63 G A F E I E I N G 13  94 R R A S N G E TL 13  12 P P P E E V E P G 12  33 C G F E A T Y L E 12  50 Y P G I E I ES R 12  66 E I E I N G Q L V 12  85 Y E K D L I E A I 12  95 R A S N G ET L E 12 105 I T N S R P P C V 12 HLA-B*5101 octamers Position 1 2 3 4 56 7 8 9 Score  83 F P Y E K D L I 25  95 R A S N G E T L 23  10 V A P PP E E V 21  18 E P G S G V R I 21  33 C G F E A T Y L 21  98 N G E T L EK I 19  19 P G S G V R I V 18  60 G G T G A F E I 18  62 T G A F E I E I18  63 G A F E I E I N 14  71 G Q L V F S K L 14  48 E Q Y P G I E I 13 67 I E I N G Q L V 13 106 T N S R P P C V 12 Class II MHC HLA-DRB1*010115-mers Position 1 2 3 4 5 6 7 8 9 Score  72 Q L V F S K L E N G G F P YE 29  37 A T Y L E L A S A V K E Q Y P 26  26 V V E Y C E P C G F E A TY L 25  63 G A F E I E I N G Q L V F S K 25  24 R I V V E Y C E P C G FE A T 24  36 E A T Y L E L A S A V K E Q Y 24  39 Y L E L A S A V K E QY P G I 24  53 I E I E S R L G G T G A F E I 24  56 E S R L G G T G A FE I E I N 24  14 P E E V E P G S G V R I V V E 23  43 A S A V K E Q Y PG I E I E S 23  20 G S G V R I V V E Y C E P C G 20  62 T G A F E I E IN G Q L V F S 20  32 P C G F E A T Y L E L A S A V 19  47 K E Q Y P G IE I E S R L G G 19  64 A F E I E I N G Q L V F S K L 19  82 G F P Y E KD L I E A I R R A 19  34 G F E A T Y L E L A S A V K E 18  54 E I E S RL G G T G A F E I E 18  90 I E A I R R A S N G E T L E K 18  99 G E T LE K I T N S R P P C V 18  31 E P C G F E A T Y L E L A S A 17  49 Q Y PG I E I E S R L G G T G 17  58 R L G G T G A F E I E I N G Q 17  66 E IE I N G Q L V F S K L E N 17  67 I E I N G Q L V F S K L E N G 17  68 EI N G Q L V F S K L E N G G 17  84 P Y E K D L I E A I R R A S N 17  86E K D L I E A I R R A S N G E 17  35 F E A T Y L E L A S A V K E Q 16 74 V F S K L E N G G F P Y E K D 16  87 K D L I E A I R R A S N G E T16  91 E A I R R A S N G E T L E K I 16   1 M S G E P G Q T S V A P P PE 15   4 E P G Q T S V A P P P E E V E 15  11 A P P P E E V E P G S G VR I 15  12 P P P E E V E P G S G V R I V 15  29 Y C E P C G F E A T Y LE L A 15   5 P G Q T S V A P P P E E V E P 14   6 G Q T S V A P P P E EV E P G 14  44 S A V K E Q Y P G I E I E S R 14  52 G I E I E S R L G GT G A F E 14  61 G T G A F E I E I N G Q L V F 13  50 Y P G I E I E S RL G G T G A 12 HLA-DRB1*0301 (DR17) 15-mers Position 1 2 3 4 5 6 7 8 9Score  64 A F E I E I N G Q L V F S K L 26  39 Y L E L A S A V K E Q Y PG I 25  72 Q L V F S K L E N G G F P Y E 23  62 T G A F E I E I N G Q LV F S 22  24 R I V V E Y C E P C G F E A T 19  71 G Q L V F S K L E N GG F P Y 19  86 E K D L I E A I R R A S N G E 19   7 Q T S V A P P P E EV E P G S 18  23 V R I V V E Y C E P C G F E A 18  50 Y P G I E I E S RL G G T G A 18  90 I E A I R R A S N G E T L E K 18  20 G S G V R I V VE Y C E P C G 17  87 K D L I E A I R R A S N G E T 17  99 G E T L E K IT N S R P P C V 16  28 E Y C E P C G F E A T Y L E L 15  37 A T Y L E LA S A V K E Q Y P 14  48 E Q Y P G I E I E S R L G G T 14  78 L E N G GF P Y E K D L I E A 14  14 P E E V E P G S G V R I V V E 13  70 N G Q LV F S K L E N G G F P 13  43 A S A V K E Q Y P G I E I E S 12  52 G I EI E S R L G G T G A F E 12  54 E I E S R L G G T G A F E I E 12  74 V FS K L E N G G F P Y E K D 12  82 G F P Y E K D L I E A I R R A 12HLA-DRB1*0401 (DR4Dw4) 15-mers Position 1 2 3 4 5 6 7 8 9 Score  36 E AT Y L E L A S A V K E Q Y 28  62 T G A F E I E I N G Q L V F S 28  86 EK D L I E A I R R A S N G E 26  87 K D L I E A I R R A S N G F T 26  90I E A I R R A S N G E T L E K 26  72 Q L V F S K L E N G G F P Y E 22 82 G F P Y E K D L I E A I R R A 22  50 Y P G I E I E S R L G G T G A20  99 G E T L E K I T N S R P P C V 20  26 V V E Y C E P C G F E A T YL 16  32 P C G F E A T Y L E L A S A V 16  47 K E Q Y P G I E I E S R LG G 16  80 N G G F P Y E K D L I E A I R 16  14 P E E V E P G S G V R IV V E 14  20 G S G V R I V V E Y C E P C G 14  22 G V R I V V E Y C E PC G F E 14  37 A T Y L E L A S A V K E Q Y P 14  39 Y L E L A S A V K EQ Y P G I 14  56 E S R L G G T G A F E I E I N 14  64 A F E I E I N G QL V F S K L 14  66 E I E I N G Q L V F S K L E N 14  10 V A P P P E E VE P G S G V R 12  12 P P P E E V E P G S G V R I V 12  16 E V E P G S GV R I V V E Y C 12  29 Y C E P C G F E A T Y L E L A 12  30 C E P C G FE A T Y L E L A S 12  31 E P C G F E A T Y L E L A S A 12  34 G F E A TY L E L A S A V K E 12  35 F E A T Y L E L A S A V K E Q 12  42 L A S AV K E Q Y P G I E I E 12  48 E Q Y P G I E I E S R L G G T 12  49 Q Y PG I E I E S R L G G T G 12  53 I E I E S R L G G T G A F E I 12  58 R LG G T G A F E I E I N G Q 12  59 L G G T G A F E I E I N G Q L 12  61 GT G A F E I E I N G Q L V F 12  63 G A F E I E I N G Q L V F S K 12  67I E I N G Q L V F S K L E N G 12  68 E I N G Q L V F S K L E N G G 12 69 I N G Q L V F S K L E N G G F 12  85 Y E K D L I E A I R R A S N G12  93 I R R A S N G E T L E K I T N 12  94 R R A S N G E T L E K I T NS 12  96 A S N G E T L E K I T N S R P 12  97 S N G E T L E K I T N S RP P 12

TABLE 2 HLA peptide motif search results User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected A1 lengthselected for subsequences to be   9 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 107 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 77 KLENGGFPY 225.000 2 16 EVEPGSGVR 90.000 3 29 YCEPCGFEA45.000 4 39 YLELASAVK 36.000 5 2 SGEPGQTSV 2.250 6 26 VVEYCEPCG 1.800 796 ASNGETLEK 1.500 8 101 TLEKITNSR 0.900 9 89 LIEAIRRAS 0.900 10 54EIESRLGGT 0.900 11 66 EIEINGQLV 0.900 12 52 GIEIESRLG 0.900 13 86EKDLIEAIR 0.500 14 42 LASAVKEQY 0.500 15 31 EPCGFEATY 0.250 16 69INGQLVFSK 0.250 17 34 GFEATYLEL 0.225 18 98 NGETLEKIT 0.225 19 61GTGAFEIEI 0.125 20 79 ENGGFPYEK 0.100 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected A1 lengthselected for subsequences to be  10 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 66 EIEInGQLVF 45.000 2 16 EVEPgSGVRI 18.000 3 29YCEPcGFEAT 9.000 4 26 VVEYcEPCGF 9.000 5 52 GIEIeSRLGG 4.500 6 2SGEPgQTSVA 2.250 7 89 LIEAiRRASN 1.800 8 20 GSGVrIVVEY 1.500 9 86EKDLiEAIRR 1.250 10 98 NGETlEKITN 1.125 11 95 RASNgETLEK 1.000 12 68EINGqLVFSK 1.000 13 54 EIESrLGGTG 0.900 14 41 ELASaVKEQY 0.500 15 100ETLEkITNSR 0.250 16 46 VKEQyPGIEI 0.225 17 39 YLELaSAVKE 0.180 18 77KLENgGFPYE 0.180 19 76 SKLEnGGFPY 0.125 20 48 EQYPgIEIES 0.075 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected A_0201 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 9 SVAPPPEEV 2.982 2 104 KITNSRPPC2.391 3 105 ITNSRPPCV 1.642 4 25 IVVEYCEPC 1.485 5 65 FEIEINGQL 1.018 647 KEQYPGIEI 0.710 7 88 DLIEAIRRA 0.703 8 59 LGGTGAFEI 0.671 9 61GTGAFEIEI 0.551 10 81 GGFPYEKDL 0.516 11 37 ATYLELASA 0.508 12 35FEATYLELA 0.501 13 15 EEVEPGSGV 0.416 14 17 VEPGSGVRI 0.345 15 97SNGETLEKI 0.315 16 70 NGQLVFSKL 0.265 17 22 GVRIVVEYC 0.205 18 45AVKEQYPGI 0.196 19 85 YEKDLIEAI 0.151 20 38 TYLELASAV 0.147 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected A_0201 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 58 RLGGtGAFEI 60.510 2 104KITNsRPPCV 33.472 3 65 FEIEiNGQLV 25.506 4 83 FPYEkDLIEA 4.502 5 33CGFEaTYLEL 3.173 6 1 MSGEpGQTSV 3.165 7 37 ATYLeLASAV 3.091 8 50YPGIeIESRL 0.641 9 69 INGQlVFSKL 0.450 10 17 VEPGsGVRIV 0.434 11 24RIVVeYCEPC 0.335 12 53 IEIEsRLGGT 0.302 13 60 GGTGaFEIEI 0.259 14 8TSVApPPEEV 0.222 15 44 SAVKeQYPGI 0.217 16 21 SGVRiVVEYC 0.201 17 55IESRlGGTGA 0.164 18 80 NGGFpYEKDL 0.139 19 81 GGFPyEKDLI 0.123 20 105ITNSrPPCVI 0.101 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected A_0205 length selected for subsequencesto be   9 scored echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 107 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 65FEIEINGQL 8.820 2 25 IVVEYCEPC 3.060 3 9 SVAPPPEEV 2.000 4 104 KITNSRPPC1.500 5 81 GGFPYEKDL 1.260 6 45 AVKEQYPGI 1.200 7 70 NGQLVFSKL 0.700 847 KEQYPGIEI 0.420 9 105 ITNSRPPCV 0.340 10 37 ATYLELASA 0.300 11 35FEATYLELA 0.252 12 17 VEPGSGVRI 0.238 13 61 GTGAFEIEI 0.200 14 97SNGETLEKI 0.150 15 30 CEPCGFEAT 0.140 16 85 YEKDLIEAI 0.126 17 51PGIEIESRL 0.105 18 59 LGGTGAFEI 0.102 19 22 GVRIVVEYC 0.100 20 15EEVEPGSGV 0.084 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected A_0205 length selected for subsequencesto be  10 scored echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 106 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 33CGFEaTYLEL 6.300 2 104 KITNsRPPCV 6.000 3 65 FEIEiNGQLV 2.520 4 53IEIEsRLGGT 1.428 5 83 FPYEkDLIEA 1.350 6 58 RLGGtGAFEI 1.200 7 69INGQlVFSKL 1.190 8 50 YPGIeIESRL 1.050 9 37 ATYLeLASAV 0.600 10 1MSGEpGQTSV 0.510 11 80 NGGFpYEKDL 0.420 12 106 TNSRpPCVIL 0.350 13 24RIVVeYCEPC 0.300 14 44 SAVKeQYPGI 0.200 15 17 VEPGsGVRIV 0.190 16 105ITNSrPPCVI 0.170 17 97 SNGEtLEKIT 0.150 18 55 IESRlGGTGA 0.119 19 60GGTGaFEIEI 0.100 20 92 AIRRaSNGET 0.100 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected A24 lengthselected for subsequences to be   9 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 107 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 34 GFEATYLEL 33.000 2 49 QYPGIEIES 11.550 3 70 NGQLVFSKL11.088 4 38 TYLELASAV 10.800 5 82 GFPYEKDLI 7.500 6 81 GGFPYEKDL 4.800 7107 NSRPPCVIL 4.800 8 75 FSKLENGGF 2.000 9 97 SNGETLEKI 1.320 10 45AVKEQYPGI 1.200 11 61 GTGAFEIEI 1.100 12 59 LGGTGAFEI 1.100 13 65FEIEINGQL 1.008 14 51 PGIEIESRL 1.008 15 106 TNSRPPCVI 1.000 16 84PYEKDLIEA 0.825 17 94 RRASNGETL 0.800 18 28 EYCEPCGPE 0.600 19 32PCGFEATYL 0.400 20 47 KEQYPGIEI 0.330 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected A24 lengthselected for subsequences to be  10 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 64 AFEIeINGQL 42.000 2 74 VFSKlENGGF 10.000 3 84PYEKdLIEAI 9.000 4 69 INGQlVFSKL 7.392 5 28 EYCEpCGFEA 6.600 6 50YPGIeIESRL 5.600 7 33 CGFEaTYLEL 5.280 8 106 TNSRpPCVIL 4.000 9 31EPCGfEATYL 4.000 10 80 NGGFpYEKDL 4.000 11 26 VVEYcEPCGF 3.000 12 66EIEInGQLVF 3.000 13 58 RLGGtGAFEI 2.200 14 56 ESRLgGTGAF 2.000 15 16EVEPgSGVRI 1.800 16 96 ASNGeTLEKI 1.650 17 105 ITNSrPPCVI 1.500 18 44SAVKeQYPGI 1.500 19 81 GGFPyEKDLI 1.200 20 60 GGTGaFEIEI 1.100 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected A3 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 77 KLENGGFPY 36.000 2 39 YLELASAVK20.000 3 101 TLEKITNSR 6.000 4 61 GTGAFEIEI 0.540 5 69 INGQLVFSK 0.360 696 ASNGETLEK 0.300 7 22 GVRIVVEYC 0.270 8 79 ENGGFPYEK 0.162 9 25IVVEYCEPC 0.135 10 45 AVKEQYPGI 0.090 11 37 ATYLELASA 0.075 12 42LASAVKEQY 0.060 13 104 KITNSRPPC 0.060 14 50 YPGIEIESR 0.060 15 72QLVFSKLEN 0.060 16 16 EVEPGSGVR 0.054 17 31 EPCGFEATY 0.054 18 9SVAPPPEEV 0.045 19 87 KDLIEAIRR 0.036 20 27 VEYCEPCGF 0.030 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected A3 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 68 EINGqLVFSK 8.100 2 58 RLGGtGAFEI2.700 3 41 ELASaVKEQY 1.800 4 78 LENGgFPYEK 0.810 5 95 RASNgETLEK 0.4006 20 GSGVrIVVEY 0.270 7 100 ETLEkITNSR 0.203 8 26 VVEYcEPCGF 0.200 9 77KLENgGFPYE 0.180 10 66 EIEInGQLVF 0.120 11 24 RIVVeYCEPC 0.090 12 104KITNsRPPCV 0.060 13 37 ATYLeLASAV 0.050 14 38 TYLElASAVK 0.045 15 83FPYEkDLIEA 0.045 16 105 ITNSrPPCVI 0.045 17 72 QLVFsKLENG 0.045 18 30CEPCgFEATY 0.036 19 22 GVRIvVEYCE 0.027 20 16 EVEPqSGVRI 0.027 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected A_1101 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 39 YLELASAVK 0.400 2 69 INGQLVFSK0.120 3 16 EVEPGSGVR 0.120 4 101 TLEKITNSR 0.080 5 61 GTGAFEIEI 0.060 650 YPGIEIESR 0.040 7 96 ASNGETLEK 0.040 8 87 KDLIEAIRR 0.036 9 77KLENGGFPY 0.036 10 79 ENGGFPYEK 0.024 11 9 SVAPPPEEV 0.020 12 45AVKEQYPGI 0.020 13 37 ATYLELASA 0.020 14 34 GFEATYLEL 0.012 15 105ITNSRPPCV 0.010 16 22 GVRIVVEYC 0.006 17 38 TYLELASAV 0.006 18 82GFPYEKDLI 0.006 19 29 YCEPCGFEA 0.006 20 73 LVFSKLENG 0.004 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected A_3101 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 101 TLEKITNSR 2.000 2 16 EVEPGSGVR0.600 3 50 YPGIEIESR 0.400 4 87 KDLIEAIRR 0.240 5 39 YLELASAVK 0.200 677 KLENGGFPY 0.180 7 37 ATYLELASA 0.060 8 69 INGQLVFSK 0.024 9 45AVKEQYPGI 0.020 10 61 GTGAFEIEI 0.020 11 9 SVAPPPEEV 0.020 12 24RIVVEYCEP 0.012 13 34 GFEATYLEL 0.012 14 73 LVFSKLENG 0.012 15 38TYLELASAV 0.012 16 105 ITNSRPPCV 0.010 17 72 QLVFSKLEN 0.008 18 82GFPYEKDLI 0.006 19 104 KITNSRPPC 0.006 20 79 ENGGFPYEK 0.006 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected A_3302 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 16 EVEPGSGVR 45.000 2 101 TLEKITNSR9.000 3 50 YPGIEIESR 3.000 4 66 EIEINGQLV 1.500 5 56 ESRLGGTGA 1.500 654 EIESRLGGT 1.500 7 68 EINGQLVFS 1.500 8 86 EKDLIEAIR 0.900 9 41ELASAVKEQ 0.900 10 88 DLIEAIRRA 0.900 11 96 ASNGETLEK 0.500 12 22GVRIVVEYC 0.500 13 1 MSGEPGQTS 0.500 14 89 LIEAIRRAS 0.500 15 107NSRPPCVIL 0.500 16 9 SVAPPPEEV 0.500 17 38 TYLELASAV 0.500 18 25IVVEYCEPC 0.500 19 45 AVKEQYPGI 0.500 20 49 QYPGIEIES 0.500 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected A_3302 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 16 EVEPGSGVR 45.000 2 101 TLEKITNSR9.000 3 50 YPGIEIESR 3.000 4 66 EIEINGQLV 1.500 5 56 ESRLGGTGA 1.500 654 EIESRLGGT 1.500 7 68 EINGQLVFS 1.500 8 86 EKDLIEAIR 0.900 9 41ELASAVKEQ 0.900 10 88 DLIEAIRRA 0.900 11 96 ASNGETLEK 0.500 12 22GVRIVVEYC 0.500 13 1 MSGEPGQTS 0.500 14 89 LIEAIRRAS 0.500 15 107NSRPPCVIL 0.500 16 9 SVAPPPEEV 0.500 17 38 TYLELASAV 0.500 18 25IVVEYCEPC 0.500 19 45 AVKEQYPGI 0.500 20 49 QYPGIEIES 0.500 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected A68.1 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 16 EVEPGSGVR 900.000 2 9 SVAPPPEEV12.000 3 50 YPGIEIESR 10.000 4 96 ASNGETLEK 9.000 5 101 TLEKITNSR 5.0006 45 AVKEQYPGI 4.000 7 79 ENGGFPYEK 3.600 8 39 YLELASAVK 3.000 9 61GTGAFEIEI 3.000 10 86 EKDLIEAIR 2.250 11 69 INGQLVFSK 1.200 12 87KDLIEAIRR 1.000 13 105 ITNSRPPCV 1.000 14 37 ATYLELASA 1.000 15 56ESRLGGTGA 0.900 16 25 IVVEYCEPC 0.800 17 73 LVFSKLENG 0.800 18 88DLIEAIRRA 0.600 19 18 EPGSGVRIV 0.600 20 26 VVEYCEPCG 0.600 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected A68.1 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 16 EVEPGSGVR 900.000 2 9 SVAPPPEEV12.000 3 50 YPGIEIESR 10.000 4 96 ASNGETLEK 9.000 5 101 TLEKITNSR 5.0006 45 AVKEQYPGI 4.000 7 79 ENGGFPYEK 3.600 8 39 YLELASAVK 3.000 9 61GTGAFEIEI 3.000 10 86 EKDLIEAIR 2.250 11 69 INGQLVFSK 1.200 12 87KDLIEAIRR 1.000 13 105 ITNSRPPCV 1.000 14 37 ATYLELASA 1.000 15 56ESRLGGTGA 0.900 16 25 IVVEYCEPC 0.800 17 73 LVFSKLKNG 0.800 18 88DLIEAIRRA 0.600 19 18 EPGSGVRIV 0.600 20 26 VVEYCIPCG 0.600 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected A68.1 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 100 ETLEkITNSR 300.000 2 16EVEPgSGVRI 18.000 3 68 EINGqLVFSK 9.000 4 15 EEVEpCSGVR 9.000 5 95RASNgETLEK 3.000 6 85 YEKDlIEAIR 2.250 7 9 SVAPpPEEVE 1.800 8 86EKDLiEAIRR 1.500 9 73 LVFSkLENGG 1.200 10 25 IVVEyCEPCG 1.200 11 105ITNSrPPCVI 1.000 12 37 ATYLeLASAV 1.000 13 78 LENGgFPYEK 0.900 14 8TSVApPPEEV 0.600 15 22 GVRIvVEYCE 0.600 16 18 EPGSgVRIVV 0.600 17 1MSGEpGQTSV 0.600 18 38 TYLElASAVK 0.600 19 49 QYPGiEIESR 0.500 20 45AVKEqYPGIE 0.400 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B14 length selected for subsequences tobe   9 scored echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 94RRASNGETL 20.000 2 57 SRLGGTGAF 5.000 3 100 ETLEKITNS 3.375 4 105ITNSRPPCV 2.000 5 88 DLIEAIRRA 1.350 6 18 EPGSGVRIV 1.200 7 70 NGQLVFSKL1.000 8 81 GGFPYEKDL 1.000 9 54 EIESRLGGT 0.900 10 97 SNGETLEKI 0.600 1191 EAIRRASNG 0.450 12 68 EINGQLVFS 0.450 13 65 FEIEINGQL 0.300 14 23VRIVVEYCE 0.300 15 21 SGVRIVVEY 0.300 16 51 PGIEIESRL 0.300 17 104KITNSRPPC 0.250 18 48 EQYPGIEIE 0.225 19 93 IRRASNGET 0.200 20 107NSRPPCVIL 0.200 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B14 length selected for subsequences tobe  10 scored echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 103EKITnSRPPC 6.750 2 33 CGFEaTYLEL 5.000 3 93 IRRAsNGETL 4.000 4 18EPGSgVRIVV 3.000 5 88 DLIEaIRRAS 2.250 6 104 KITNsRPPCV 2.000 7 106TNSRpPCVIL 1.000 8 50 YPGIeIESRL 1.000 9 69 INGQlVFSKL 1.000 10 37ATYLeLASAV 1.000 11 31 EPCGfEATYL 0.900 12 48 EQYPgIEIES 0.750 13 76SKLEnGGFPY 0.750 14 83 FPYEkDLIEA 0.750 15 8 TSVApPPEEV 0.600 16 96ASNGeTLEKI 0.600 17 44 SAVKeQYPGI 0.600 18 57 SRLGgTGAFE 0.500 19 53IEIEsRLGGT 0.450 20 21 SGVRiVVEYC 0.300 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_2705 lengthselected for subsequences to be   9 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 107 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 94 RRASNGETL 6000.000 2 57 SRLGGTGAF 1000.000 3 93IRRASNGET 200.000 4 27 VEYCEPCGF 75.000 5 77 KLENGGFPY 45.000 6 39YLELASAVK 30.000 7 65 FEIEINGQL 30.000 8 47 KEQYFGIEI 27.000 9 69INGQLVFSK 20.000 10 23 VRIVVEYCE 20.000 11 101 TLEKITNSR 15.000 12 67IEINGQLVF 15.000 13 107 NSRPPCVIL 10.000 14 96 ASNGETLEK 10.000 15 85YEKDLIEAI 9.000 16 17 VEPGSGVRI 9.000 17 81 GGFPYEKDL 7.500 18 106TNSRPPCVI 6.000 19 97 SNGETLEKI 6.000 20 75 FSKLENGGF 5.000 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected B_2705 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 93 IRRAsNGETL 2000.000 2 94RRASnGETLE 60.000 3 78 LENGgFPYEK 30.000 4 95 RASNgETLEK 30.000 5 58RLGGtGAFEI 27.000 6 33 CGFEaTYLEL 25.000 7 106 TNSRpPCVIL 20.000 8 71GQLVfSKLEN 20.000 9 23 VRIVvEYCEP 20.000 10 57 SRLGgTGAFE 20.000 11 69INGQlVFSKL 20.000 12 30 CEPCgFEATY 15.000 13 85 YEKDlIEAIR 15.000 14 37ATYLeLASAV 15.000 15 48 EQYPgIEIES 10.000 16 50 YPGIeIESRL 10.000 17 104KITNsRPPCV 9.000 18 65 FEIEiNGQLV 9.000 19 81 GGFPyEKDLI 7.500 20 83FPYEkDLIEA 5.000 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B_3501 length selected for subsequencesto be   9 scored echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 107 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 31EPCGFEATY 40.000 2 75 FSKLENGGF 22.500 3 107 NSRPPCVIL 15.000 4 42LASAVKEQY 6.000 5 18 EPGSGVRIV 4.000 6 45 AVKEQYPGI 2.400 7 21 SGVRIVVEY2.000 8 56 ESRLGGTGA 1.500 9 77 KLENGGFPY 1.200 10 81 GGFPYEKDL 1.000 111 MSGEPGQTS 1.000 12 70 NGQLVFSKL 1.000 13 97 SNGETLEKI 0.800 14 83FPYEKDLIE 0.400 15 61 GTGAFEIEI 0.400 16 59 LGGTGAFEI 0.400 17 106TNSRPPCVI 0.400 18 50 YPGIEIESR 0.300 19 22 GVRIVVEYC 0.300 20 11APPPEEVEP 0.300 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B_3501 length selected for subsequencesto be  10 scored echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 106 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 31EPCGfEATYL 30.000 2 50 YPGIeIESRL 20.000 3 56 ESRLgGTGAF 15.000 4 20GSGVrIVVEY 10.000 5 83 FPYEkDLIEA 6.000 6 18 EPGSgVRIVV 4.000 7 33CGFEaTYLEL 2.000 8 1 MSGEpGQTSV 2.000 9 96 ASNGeTLEKI 2.000 10 41ELASaVKEQY 2.000 11 44 SAVKeQYPGI 1.200 12 69 INGQlVFSKL 1.000 13 8TSVApPPEEV 1.000 14 80 NGGFpYEKDL 1.000 15 106 TNSRpPCVIL 1.000 16 58RLGGtGAFEI 0.800 17 81 GGFPyEKDLI 0.600 18 26 VVEYcEPCGF 0.450 19 36EATYlELASA 0.450 20 21 PPPEeVEPGS 0.400 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber ot results requested  20 HLA molecule type selected B_3901 lengthselected for subsequences to be   9 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 107 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 94 RRASNGETL 15.000 2 34 GFEATYLEL 9.000 3 38 TYLELASAV4.000 4 66 EIEINGQLV 3.000 5 2 SGEPGQTSV 3.000 6 97 SNGETLEKI 3.000 7 70NGQLVFSKL 3.000 8 81 GGFPYEKDL 3.000 9 18 EPGSGVRIV 1.500 10 65FEIEINGQL 1.200 11 57 SRLGGTGAF 1.000 12 106 TNSRPPCVI 1.000 13 9SVAPPPEEV 1.000 14 59 LGGTGAFEI 1.000 15 105 ITNSRPPCV 1.000 16 107NSRPPCVIL 0.900 17 45 AVKEQYPGI 0.600 18 51 PGIEIESRL 0.600 19 88DLIEAIRRA 0.600 20 100 ETLEKITNS 0.600 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_3901 lengthselected for subsequences to be  10 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 33 CGFEaTYLEL 12.000 2 64 AFEIeINGQL 9.000 3 93IRRAsNGETL 4.500 4 46 VKEQyPGIEI 3.000 5 16 EVEPgSGVRI 3.000 6 106TNSRpPCVIL 3.000 7 69 INGQlVFSKL 3.000 8 31 EPCGfEATYL 3.000 9 44SAVKeQYPGI 2.000 10 1 MSGEpGQTSV 2.000 11 8 TSVApPPEEV 2.000 12 37ATYLeLASAV 2.000 13 80 NGGFpYEKDL 1.500 14 50 YPGIeIESRL 1.500 15 96ASNGeTLEKI 1.500 16 58 RLGGtGAFEI 1.000 17 105 ITNSrPPCVI 1.000 18 81GGFPyEKDLI 1.000 19 104 KITNsRPPCV 1.000 20 83 FPYEkDLIEA 0.600 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected B40 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 65 FEIEINGQL 80.000 2 3 GEPGQTSVA40.000 3 35 FEATYLELA 40.000 4 15 EEVEPGSGV 24.000 5 67 IEINGQLVF 16.0006 81 GGFPYEKDL 8.000 7 27 VEYCEPCGF 8.000 8 47 KEQYPGIEI 6.000 9 17VEPGSGVRI 4.000 10 30 CEPCGFEAT 4.000 11 99 GETLEKITN 2.400 12 90IEAIRRASN 2.400 13 37 ATYLELASA 2.000 14 85 YEKDLIEAI 2.000 15 53IEIESRLGG 1.600 16 40 LELASAVKE 0.800 17 107 NSRPPCVIL 0.750 18 29YCEPCGFEA 0.500 19 70 NGQLVFSKL 0.500 20 78 LENGGFPYE 0.400 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected B40 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 55 IESRlGGTGA 20.000 2 53IEIEsRLGGT 16.000 3 65 FEIEiNGQLV 16.000 4 67 IEINgQLVFS 16.000 5 99GETLeKITNS 8.000 6 35 FEATyLELAS 8.000 7 87 KDLIeAIRRA 5.000 8 17VEPGsGVRIV 4.000 9 30 CEPCgFEATY 4.000 10 33 CGFEaTYLEL 2.000 11 15EEVEpGSGVR 1.600 12 81 GGFPyEKDLI 1.600 13 27 VEYCePCGFE 1.200 14 83FPYEkDLIEA 1.000 15 40 LELAsAVKEQ 0.800 16 3 GEPGqTSVAP 0.800 17 90IEAIrRASNG 0.800 18 106 TNSRpPCVIL 0.750 19 8 TSVApPPEEV 0.600 20 2SGEPgQTSVA 0.500 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B_5201 length selected for subsequencesto be   9 scored echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 107 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 18EPGSGVRIV 75.000 2 67 IEINGQLVF 22.500 3 59 LGGTGAFEI 11.250 4 98NGETLEKIT 11.000 5 19 PGSGVRIVV 10.000 6 106 TNSRPPCVI 10.000 7 48EQYPGIEIE 9.900 8 2 SGEPGQTSV 9.000 9 81 GGFPYEKDL 6.600 10 38 TYLELASAV4.800 11 27 VEYCEPCGF 3.750 12 83 FPYEKDLIE 3.000 13 17 VEPGSGVRI 3.00014 70 NGQLVFSKL 2.400 15 85 YEKDLIEAI 2.200 16 3 GEPGQTSVA 2.200 17 82GFPYEKDLI 2.200 18 97 SNGETLEKI 2.178 19 61 GTGAFEIEI 1.800 20 105ITNSRPPCV 1.500 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B_5201 length selected for subsequencesto be  10 scored echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 106 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 18EPGSgVRIVV 100.000 2 17 VEPGsGVRIV 45.000 3 81 GGFPyEKDLI 33.000 4 105ITNSrPPCVI 15.000 5 37 ATYLeLASAV 12.000 6 66 EIEInGQLVF 9.000 7 33CGFEaTYLEL 9.000 8 60 GGTGaFEIEI 7.500 9 2 SGEPgQTSVA 6.600 10 83FPYEkDLIEA 3.300 11 1 MSGEpGQTSV 2.700 12 97 SNGEtLEKIT 2.640 13 65FEIEiNGQLV 2.640 14 50 YPGIeIESRL 2.400 15 48 EQYPgIEIES 2.400 16 106TNSRpPCVIL 2.000 17 96 ASNGeTLEKI 1.815 18 58 RLGGtGAFEI 1.500 19 8TSVApPPEEV 1.320 20 59 LGGTgAFEIE 1.238 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B60 lengthselected for subsequences to be   9 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 107 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 65 FEIEINGQL 387.200 2 17 VEPGSGVRI 17.600 3 15 EEVEPGSGV16.000 4 47 KEQYPGIEI 16.000 5 85 YEKDLIEAI 8.800 6 107 NSRPPCVIL 8.0007 35 FEATYLELA 8.000 8 70 NGQLVFSKL 4.840 9 3 GEPGQTSVA 4.000 10 81GGFPYEKDL 4.000 11 30 CEPCGFEAT 4.000 12 67 IEINGQLVF 3.200 13 90IEAIRRASN 2.400 14 99 GETLEEKITN 2.400 15 40 LELASAVKE 1.760 16 53IEIESRLGG 1.600 17 51 PGIEIESRL 0.968 18 55 IESRLGGTG 0.880 19 34GFEATYLEL 0.800 20 94 RRASNGETL 0.800 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B60 lengthselected for subsequences to be  10 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 65 FEIEiNGQLV 16.000 2 106 TNSRpPCVIL 16.000 3 53IEIEsRLGGT 8.000 4 33 CGFEaTYLEL 8.000 5 17 VEPGsGVRIV 8.000 6 55IESRlGGTGA 8.000 7 69 INGQlVFSKL 4.840 8 50 YPGIeIESRL 4.840 9 80NGGFpYEKDL 4.000 10 31 EPCGfEATYL 4.000 11 35 FEATyLELAS 3.520 12 67IEINgQLVFS 3.200 13 87 KDLIeAIRRA 1.100 14 78 LENGgFPYEK 0.800 15 15EEVEpGSGVR 0.800 16 99 GETLeKITNS 0.800 17 30 CEPCgFEATY 0.800 18 90IEAIrRASNG 0.800 19 3 GEPGqTSVAP 0.800 20 40 LELAsAVKEQ 0.800 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected B61 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 15 EEVEPGSGV 80.000 2 35 FEATYLELA40.000 3 3 GEPGQTSVA 22.000 4 65 FELEINGQL 16.000 5 85 YEKDLIEAI 16.0006 17 VEPGSGVRI 8.000 7 47 KEQYPGIEI 8.000 8 30 CEPCGFEAT 4.000 9 99GETLEKITN 2.640 10 90 IEAIRRASN 2.400 11 27 VEYCEPCGF 1.600 12 67IEINGQLVF 1.600 13 2 SGEPGQTSV 1.000 14 18 EPGSGVRIV 1.000 15 105ITNSRPPCV 1.000 16 37 ATYLELASA 1.000 17 53 IEIESRLGG 0.800 18 40LELASAVKE 0.800 19 81 GGFPYEKDL 0.660 20 29 YCEPCGFEA 0.500 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected B61 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 65 FEIEiNGQLV 80.000 2 17VEPGsGVRIV 40.000 3 55 IESRlGGTGA 20.000 4 87 KDLIeAIRRA 10.000 5 53IEIEsRLGGT 8.000 6 14 PEEVePGSGV 4.000 7 99 GETLeKITNS 3.520 8 37ATYLeLASAV 2.000 9 8 TSVApPPEEV 2.000 10 67 IEINgQLVFS 1.600 11 35FEATyLELAS 1.600 12 1 MSGEpGQTSV 1.000 13 18 EPGSgVRIVV 1.000 14 36EATYlELASA 1.000 15 83 FPYEkDLIEA 1.000 16 15 EEVEpGSGVR 0.800 17 27VEYCePCGFE 0.800 18 30 CEPCgFEATY 0.800 19 90 IEAIrRASNG 0.800 20 40LELAsAVKEQ 0.800 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B62 length selected for subsequences tobe   9 scored echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 107 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 77KLENGGFPY 24.000 2 21 SGVRIVVEY 4.800 3 75 FSKLENGGF 3.000 4 31EPCGFEATY 2.640 5 88 DLIEAIRRA 2.200 6 42 LASAVKEQY 2.000 7 48 EQYPGIEIE0.960 8 71 GQLVFSKLE 0.800 9 6 GQTSVAPPP 0.800 10 67 IEINGQLVF 0.686 1122 GVRIVVEYC 0.660 12 58 RLGGTGAFE 0.480 13 57 SRLGGTGAF 0.480 14 18EPGSGVRIV 0.400 15 59 LGGTGAFEI 0.400 16 56 ESRLGGTGA 0.360 17 45AVKEQYPGI 0.330 18 104 KITNSRPPC 0.250 19 72 QLVFSKLEN 0.240 20 61GTGAFEIEI 0.240 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B62 length selected for subsequences tobe  10 scored echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 41ELASaVKEQY 40.000 2 58 RLGGtGAFEI 9.600 3 66 EIEInGQLVF 7.920 4 56ESRLgGTGAF 6.000 5 20 GSGVrIVVEY 4.800 6 92 AIRRaSNGET 1.500 7 48EQYPgIEIES 1.152 8 26 VVEYcEPCGF 0.600 9 24 RIVVeYCEPC 0.500 10 104KITNsRPPCV 0.500 11 71 GQLVfSKLEN 0.480 12 76 SKLEnGGFPY 0.440 13 88DLIEaIRRAS 0.440 14 6 GQTSvAPPPE 0.400 15 1 MSGEpGQTSV 0.264 16 18EPGSgVRIVV 0.264 17 69 INGQlVFSKL 0.260 18 21 SGVRiVVEYC 0.220 19 30CEPCgFEATY 0.220 20 74 VFSKlENGGF 0.200 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B7 lengthselected for subsequences to be   9 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 107 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 107 NSRPPCVIL 60.000 2 45 AVKEQYPGI 6.000 3 22 GVRIVVEYC5.000 4 70 NGQLVFSKL 4.000 5 81 GGFPYEKDL 4.000 6 18 EPGSGVRIV 4.000 7 9SVAPPPEEV 1.500 8 56 ESRLGGTGA 1.000 9 106 TNSRPPCVI 0.600 10 11APPPEEVEP 0.600 11 25 IVVEYCEPC 0.500 12 65 FEIEINGQL 0.400 13 61GTGAFEIEI 0.400 14 31 EPCGFEATY 0.400 15 94 RRASNGETL 0.400 16 59LGGTGAFEI 0.400 17 51 PGIEIESRL 0.400 18 32 PCGFEATYL 0.400 19 97SNGETLEKI 0.400 20 92 AIRRASNGE 0.300 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B7 lengthselected for subsequences to be  10 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 50 YPGIeIESRL 80.000 2 31 EPCGfEATYL 80.000 3 18EPGSgVRIVV 6.000 4 106 TNSRpPCVIL 6.000 5 80 NGGFpYEKDL 4.000 6 69INGQlVFSKL 4.000 7 93 IRRAsNGETL 4.000 8 33 CGFEaTYLEL 4.000 9 92AIRRaSNGET 3.000 10 83 FPYEkDLIEA 2.000 11 44 SAVKeQYPGI 1.200 12 96ASNGeTLEKI 1.200 13 11 APPPeEVEPG 0.600 14 16 EVEPgSGVRI 0.600 15 37ATYLeLASAV 0.600 16 105 ITNSrPPCVI 0.600 17 22 GVRIvVEYCE 0.500 18 60GGTGaFEIEI 0.400 19 81 GGFPyEKDLI 0.400 20 58 RLGGtGAFEI 0.400 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected B8 length selected for subsequences to be   8 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 108 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 83 FPYEKDLI 6.000 2 107 NSRPPCVI1.000 3 91 EAIRRASN 0.800 4 20 GSGVRIVV 0.600 5 18 EPGSGVRI 0.400 6 95RASNGETL 0.400 7 100 ETLEKITN 0.300 8 105 ITNSRPPC 0.200 9 10 VAPPPEEV0.120 10 73 LVFSKLEN 0.100 11 43 ASAVKEQY 0.100 12 22 GVRIVVEY 0.100 1336 EATYLELA 0.080 14 31 EPCGFEAT 0.080 15 66 EIEINGQL 0.080 16 4EPGQTSVA 0.080 17 33 CGFEATYL 0.060 18 71 GQLVFSKL 0.060 19 56 ESRLGGTG0.040 20 106 TNSRPPCV 0.030 User Parameters and Scoring Informationmethod selected to limit number of results explicit number number ofresults requested  20 HLA molecule type selected B8 length selected forsubsequences to be   8 scored echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence115 number of subsequence scores calculated 108 number of top-scoringsubsequences reported  20 back in scoring output table Scoring ResultsScore (Estimate of Half Time of Disassociation of a Molecule StartSubsequence Residue Containing Rank Position Listing This Subsequence) 183 FPYEKDLI 6.000 2 107 NSRPPCVI 1.000 3 91 EAIRRASN 0.800 4 20 GSGVRIVV0.600 5 18 EPGSGVRI 0.400 6 95 RASNGETL 0.400 7 100 ETLEKITN 0.300 8 105ITNSRPPC 0.200 9 10 VAPPPEEV 0.120 10 73 LVFSKLEN 0.100 11 43 ASAVKEQY0.100 12 22 GVRIVVEY 0.100 13 36 EATYLELA 0.080 14 31 EPCGFEAT 0.080 1566 EIEINGQL 0.080 16 4 EPGQTSVA 0.080 17 33 CGFEATYL 0.060 18 71GQLVFSKL 0.060 19 56 ESRLGGTG 0.040 20 106 TNSRPPCV 0.030 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected Cw_0702 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 20 GSGVrIVVEY 38.400 2 30CEPCgFEATY 16.000 3 41 ELASaVKEQY 16.000 4 50 YPGIeIESRL 7.920 5 76SKLEnGGFPY 4.000 6 69 INGQlVFSKL 2.880 7 18 EPGSgVRIVV 2.400 8 33CGFEaTYLEL 1.440 9 80 NGGFpYEKDL 1.440 10 56 ESRLgGTGAF 1.200 11 93IRRAsNGETL 1.200 12 64 AFEIeINGQL 1.200 13 66 EIEInGQLVF 1.000 14 35FEATyLELAS 0.960 15 87 KDLIeAIRRA 0.800 16 97 SNGEtLEKIT 0.800 17 17VEPGsGVRIV 0.800 18 21 SGVRiVVEYC 0.800 19 28 EYCEpCGFEA 0.720 20 48EQYPgIEIES 0.672 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B8 length selected for subsequences to be 10 scored echoing mode selected for input sequence Y echoing formatnumbered lines length of user's input peptide sequence 115 number ofsubsequence scores calculated 106 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 50YPGIeIESRL 0.800 2 93 IRRAsNGETL 0.400 3 31 EPCGfEATYL 0.320 4 104KITNsRPPCV 0.300 5 18 EPGSgVRIVV 0.240 6 56 ESRLgGTGAF 0.200 7 44SAVKeQYPGI 0.200 8 92 AIRRaSNGET 0.200 9 69 INGQlVFSKL 0.200 10 106TNSRpPCVIL 0.200 11 42 LASAvKEQYP 0.160 12 33 CGFEaTYLEL 0.060 13 105ITNSrPPCVI 0.050 14 58 RLGGtGAFEI 0.050 15 96 ASNGeTLEKI 0.050 16 1MSGEpGQTSV 0.045 17 75 FSKLeNGGFP 0.040 18 80 NGGFpYEKDL 0.040 19 72QLVFsKLENG 0.040 20 53 IEIEsRLGGT 0.030 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_2702 lengthselected for subsequences to be   9 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 107 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 57 SRLGGTGAF 200.000 2 94 RRASNGETL 180.000 3 93IRRASNGET 20.000 4 27 VEYCEPCGF 15.000 5 77 KLENGGFPY 9.000 6 67IEINGQLVF 3.000 7 47 KEQYPGIEI 2.700 8 23 VRIVVEYCE 2.000 9 42 LASAVKEQY1.000 10 75 FSKLENGGF 1.000 11 85 YEKDLIEAI 0.900 12 17 VEPGSGVRI 0.90013 65 FEIEINGQL 0.900 14 97 SNGETLEKI 0.600 15 106 TNSRPPCVI 0.600 16 37ATYLELASA 0.500 17 21 SGVRIVVEY 0.500 18 107 NSRPPCVIL 0.300 19 30CEPCGFEAT 0.300 20 48 EQYPGIEIE 0.300 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_2702 lengthselected for subsequences to be  10 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 93 IRRAsNGETL 60.000 2 94 RRASnGETLE 6.000 3 30CEPCgFEATY 3.000 4 58 RLGGtGAFEI 2.700 5 23 VRIVvEYCEP 2.000 6 57SRLGgTGAFE 2.000 7 48 EQYPgIEIES 1.500 8 26 VVEYcEPCGF 1.000 9 20GSGVrIVVEY 1.000 10 71 GQLVfSKLEN 1.000 11 41 ELASaVKEQY 0.900 12 33CGFEaTYLEL 0.750 13 81 GGFPyEKDLI 0.750 14 106 TNSRpPCVIL 0.600 15 69INGQlVFSKL 0.600 16 83 FPYEkDLIEA 0.500 17 37 ATYLeLASAV 0.500 18 55IESRlGGTGA 0.300 19 96 ASNGeTLEKI 0.300 20 56 ESRLgGTGAF 0.300 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected B_3701 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 65 FEIEiNGQLV 10.000 2 67IEINgQLVFS 5.000 3 81 GGFPyEKDLI 5.000 4 87 KDLIeAIRRA 4.000 5 30CEPCgFEATY 2.000 6 17 VEPGsGVRIV 2.000 7 50 YPGIeIESRL 1.500 8 64AFEIeINGQL 1.500 9 69 INGQlVFSKL 1.500 10 99 GETLeKITNS 1.000 11 60GGTGaFEIEI 1.000 12 46 VKEQyPGIEI 1.000 13 53 IEIEsRLGGT 1.000 14 16EVEPgSGVRI 1.000 15 44 SAVKeQYPGI 1.000 16 105 ITNSrPPCVI 1.000 17 96ASNGeTLEKI 1.000 18 80 NGGFpYEKDL 1.000 19 55 IESRlGGTGA 1.000 20 31EPCGfEATYL 1.000 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B_3801 length selected for subsequencesto be   9 scored echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 107 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 34GFEATYLEL 6.000 2 70 NGQLVFSKL 1.560 3 38 TYLELASAV 1.040 4 81 GGFPYEKDL1.000 5 97 SNGETLEKI 0.720 6 66 EIEINGQLV 0.600 7 2 SGEPGQTSV 0.600 8 82GFPYEKDLI 0.600 9 49 QYPGIEIES 0.520 10 18 EPGSGVRIV 0.400 11 31EPCGFEATY 0.400 12 89 LIEAIRRAS 0.390 13 98 NGETLEKIT 0.390 14 77KLENGGFPY 0.300 15 61 GTGAFEIEI 0.300 16 107 NSRPPCVIL 0.300 17 75FSKLENGGF 0.300 18 106 TNSRPPCVI 0.300 19 29 YCEPCGFEA 0.300 20 54EIESRLGGT 0.300 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B_3801 length selected for subsequencesto be  10 scored echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 106 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 64AFEIeINGQL 7.800 2 31 EPCGfEATYL 4.800 3 66 ETEInGQLVF 3.000 4 26VVEYcEPCGF 3.000 5 50 YPGIeIESRL 2.600 6 74 VFSKlENGGF 2.000 7 33CGFEaTYLEL 2.000 8 69 INGQlVFSKL 1.560 9 106 TNSRpPCVIL 1.000 10 80NGGFpYEKDL 1.000 11 16 EVEPgSGVRI 0.900 12 96 ASNGeTLEKI 0.720 13 34GFEAtYLELA 0.600 14 60 GGTGaFEIEI 0.600 15 58 RLGGtGAFEI 0.600 16 18EPGSgVRIVV 0.520 17 83 FPYEkDLIEA 0.400 18 28 EYCEpCGFEA 0.400 19 1MSGEpGQTSV 0.400 20 2 SGEPgQTSVA 0.300 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_3902 lengthselected for subsequences to be   9 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 107 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 70 NGQLVFSKL 2.400 2 81 GGFPYEKDL 2.400 3 94 RRASNGETL2.000 4 34 GFEATYLEL 2.000 5 107 NSRPPCVIL 0.600 6 57 SRLGGTGAF 0.500 765 FEIEINGQL 0.480 8 51 PGIEIESRL 0.240 9 32 PCGFEATYL 0.200 10 75FSKLENGGF 0.150 11 86 EKDLIEAIR 0.120 12 6 GQTSVAPPP 0.120 13 71GQLVFSKLE 0.120 14 46 VKEQYPGIE 0.120 15 89 LIEAIRRAS 0.120 16 21SGVRIVVEY 0.120 17 98 NGETLEKIT 0.120 18 36 EATYLELAS 0.120 19 38TYLELASAV 0.120 20 31 EPCGFEATY 0.120 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_3902 lengthselected for subsequences to be   9 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 107 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 70 NGQLVFSKL 2.400 2 81 GGFPYEKDL 2.400 3 94 RRASNGETL2.000 4 34 GFEATYLEL 2.000 5 107 NSRPPCVIL 0.600 6 57 SRLGGTGAF 0.500 765 FEIEINGQL 0.480 8 51 PGIEIESRL 0.240 9 32 PCGFEATYL 0.200 10 75FSKLENGGF 0.150 11 86 EKDLIEAIR 0.120 12 6 GQTSVAPPP 0.120 13 71GQLVFSKLE 0.120 14 46 VKEQYPGIE 0.120 15 89 LIEAIRRAS 0.120 16 21SGVRIVVEY 0.120 17 98 NGETLEKIT 0.120 18 36 EATYLELAS 0.120 19 38TYLELASAV 0.120 20 31 EPCGFEATY 0.120 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_3902 lengthselected for subsequences to be  10 scored echoing mode selected forinput sequence Y echoing format numbered lines lengh of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 69 INGQlVFSKL 2.400 2 64 AFEIeINGQL 2.400 3 50 YPGIeIESRL2.400 4 80 NGGFpYEKDL 2.400 5 106 TNSRpPCVIL 2.000 6 31 EPCGfEATYL 2.0007 33 CGFEaTYLEL 2.000 8 48 EQYPgIEIES 1.200 9 76 SKLEnGGFPY 1.000 10 71GQLVfSKLEN 1.000 11 46 VKEQyPGIEI 1.000 12 103 EKITnSRPPC 1.000 13 93IRRAsNGETL 0.600 14 66 EIEInGQLVF 0.500 15 26 VVEYcEPCGF 0.500 16 74VFSKlENGGF 0.500 17 56 ESRLgGTGAF 0.150 18 24 RIVVeYCEPC 0.120 19 34GFEAtYLELA 0.120 20 60 GGTGaFEIEI 0.120 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_4403 lengthselected for subsequences to be   9 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 107 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 67 IEINGQLVF 200.000 2 27 VEYCEPCGF 40.000 3 21 SGVRIVVEY36.000 4 65 FEIEINGQL 20.000 5 35 FEATYLELA 12.000 6 3 GEPGQTSVA 9.000 715 EEVEPGSGV 8.000 8 17 VEPGSGVRI 6.000 9 42 LASAVKEQY 4.500 10 31EPCGFEATY 4.500 11 85 YEKDLIEAI 4.000 12 30 CEPCGFEAT 4.000 13 47KEQYPGIEI 4.000 14 90 IEAIRRASN 3.600 15 53 IEIESRLGG 2.000 16 40LELASAVKE 1.800 17 99 GETLEKITN 1.200 18 75 FSKLENGGF 1.000 19 57SRLGGTGAF 0.900 20 78 LENGGFPYE 0.600 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_4403 lengthselected for subsequences to be  10 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 30 CEPCgFEATY 120.000 2 53 IEIEsRLGGT 30.000 3 67IEINgQLVFS 30.000 4 65 FEIEiNGQLV 20.000 5 17 VEPGsGVRIV 18.000 6 20GSGVrIVVEY 9.000 7 99 GETLeKITNS 9.000 8 35 FEATyLELAS 8.000 9 55IESRlGGTGA 6.000 10 40 LELAsAVKEQ 5.400 11 87 KDLIeAIRRA 2.250 12 76SKLEnGGFPY 1.800 13 90 IEAIrRASNG 1.800 14 21 SGVRiVVEYC 1.800 15 56ESRLgGTGAF 1.500 16 41 ELASaVKEQY 0.900 17 15 EEVEpGSGVR 0.800 18 96ASNGeTLEKI 0.675 19 3 GEPGqTSVAP 0.600 20 78 LENGgFPYEK 0.600 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected B_5101 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 18 EPGSGVRIV 484.000 2 59 LGGTGAFEI114.400 3 2 SGEPGQTSV 48.400 4 81 GGFPYEKDL 44.000 5 70 NGQLVFSKL 22.0006 31 EPCGFEATY 7.260 7 97 SNGETLEKI 5.856 8 36 EATYLELAS 5.000 9 19PGSGVRIVV 4.840 10 66 EIEINGQLV 4.840 11 45 AVKEQYPGI 4.400 12 82GFPYEKDLI 4.400 13 61 GTGAFEIEI 4.000 14 106 TNSRPPCVI 4.000 15 83FPYEKDLIE 2.860 16 105 ITNSRPPCV 2.600 17 42 LASAVKEQY 2.595 18 51PGIEIESRL 2.420 19 4 EPGQTSVAP 2.200 20 9 SVAPPPEEV 2.200 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected B_5101 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 18 EPGSgVRIVV 440.000 2 44SAVKeQYPGI 220.000 3 31 EPCGfEATYL 220.000 4 81 GGFPyEKDLI 176.000 5 50YPGIeIESRL 157.300 6 60 GGTGaFEIEI 88.000 7 33 CGFEaTYLEL 48.400 8 83FPYEkDLIEA 31.460 9 80 NGGFpYEKDL 22.000 10 36 EATYlELASA 11.000 11 16EVEPgSGVRI 8.800 12 96 ASNGeTLEKI 5.856 13 105 ITNSrPPCVI 5.200 14 37ATYLeLASAV 4.000 15 1 MSGEpGQTSV 3.461 16 21 SGVRiVVEYC 2.420 17 58RLGGtGAFEI 2.420 18 4 EPGQtSVAPP 2.200 19 8 TSVApPPEEV 2.200 20 2SGEPgQTSVA 2.000 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B_5102 length selected for subsequencesto be   9 scored echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 107 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 18EPGSGVRIV 242.000 2 81 GGFPYEKDL 110.000 3 59 LGGTGAFEI 96.800 4 70NGQLVFSKL 48.400 5 2 SGEPGQTSV 24.200 6 51 PGIEIESRL 13.200 7 83FPYEKDLIE 11.000 8 97 SNGETLEKI 10.648 9 38 TYLELASAV 6.600 10 19PGSGVRIVV 4.840 11 106 TNSRPPCVI 4.400 12 61 GTGAFEIEI 4.000 13 82GFPYEKDLI 4.000 14 31 EPCGFEATY 3.630 15 63 GAFEIEING 2.750 16 36EATYLELAS 2.500 17 50 YPGIEIESR 2.420 18 45 AVKEQYPGI 2.420 19 9SVAPPPEEV 2.200 20 105 ITNSRPPCV 2.000 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_5102 lengthselected for subsequences to be  10 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 44 SAVKeQYPGI 726.000 2 50 YPGIeIESRL 400.000 3 81GGFPyEKDLI 400.000 4 18 EPGSgVRIVV 220.000 5 31 EPCGfEATYL 121.000 6 33CGFEaTYLEL 121.000 7 83 FPYEkDLIEA 110.000 8 60 GGTGaFEIEI 88.000 9 80NGGFpYEKDL 22.000 10 37 ATYLeLASAV 11.000 11 96 ASNGeTLEKI 10.648 12 21SGVRiVVEYC 8.785 13 8 TSVApPPEEV 6.600 14 36 EATYlELASA 5.000 15 58RLGGtGAFEI 4.840 16 16 EVEPgSGVRI 4.000 17 105 ITNSrPPCVI 4.000 18 65FEIEiNGQLV 3.194 19 63 GAFEiEINGQ 3.025 20 1 MSGEpGQTSV 2.662 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected B_5103 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 44 SAVKeQYPGI 110.000 2 81GGFPyEKDLI 52.800 3 18 EPGSgVRIVV 44.000 4 60 GGTGaFEIEI 44.000 5 33CGFEaTYLEL 7.920 6 37 ATYLeLASAV 6.600 7 31 EPCGfEATYL 6.600 8 83FPYEkDLIEA 6.600 9 80 NGGFpYEKDL 6.000 10 50 YPGIeIESRL 6.000 11 36EATYlELASA 5.000 12 21 SGVRiVVEYC 2.420 13 2 SGEPgQTSVA 2.420 14 1MSGEpGQTSV 2.420 15 104 KITNsRPPCV 2.420 16 58 RLGGtGAFEI 2.420 17 96ASNGeTLEKI 2.200 18 8 TSVApPPEEV 2.200 19 16 EVEPgSGVRI 2.200 20 105ITNSrPPCVI 2.000 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected B_5103 length selected for subsequencesto be  10 scored echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 106 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 44SAVKeQYPGI 110.000 2 81 GGFPyEKDLI 52.800 3 18 EPGSgVRIVV 44.000 4 60GGTGaFEIEI 44.000 5 33 CGFEaTYLEL 7.920 6 37 ATYLeLASAV 6.600 7 31EPCGfEATYL 6.600 8 83 FPYEkDLIEA 6.600 9 80 NGGFpYEKDL 6.000 10 50YPGIeIESRL 6.000 11 36 EATYlELASA 6.000 12 21 SGVRiVVEYC 5.000 13 2SGEPgQTSVA 2.420 14 1 MSGEpGQTSV 2.420 15 104 KITNsRPPCV 2.420 16 58RLGGtGAFEI 2.420 17 96 ASNGeTLEKI 2.200 18 8 TSVApPPEEV 2.200 19 16EVEPgSGVRI 2.200 20 105 ITNSrPPCVI 2.000 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_5801 lengthselected for subsequences to be   9 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 107 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 75 FSKLENGGF 40.000 2 42 LASAVKEQY 4.500 3 107 NSRPPCVIL4.000 4 61 GTGAFEIEI 3.000 5 105 ITNSRPPCV 3.000 6 37 ATYLELASA 2.400 71 MSGEPGQTS 0.880 8 67 IEINGQLVF 0.660 9 56 ESRLGGTGA 0.600 10 21SGVRIVVEY 0.540 11 27 VEYCEPCGF 0.400 12 63 GAFEIEING 0.330 13 100ETLEKITNS 0.317 14 95 RASNGETLE 0.300 15 20 GSGVRIVVE 0.240 16 96ASNGETLEK 0.220 17 44 SAVKEQYPG 0.220 18 2 SGEPGQTSV 0.200 19 10VAPPPEEVE 0.200 20 57 SRLGGTGAF 0.200 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected B_5801 lengthselected for subsequences to be  10 scored echoing mode selected forinput sequence Y echoing format numbered lines length of user's inputpeptide sequence 115 number of subsequence scores calculated 106 numberof top-scoring subsequences reported  20 back in scoring output tableScoring Results Score (Estimate of Half Time of Disassociation of aMolecule Start Subsequence Residue Containing Rank Position Listing ThisSubsequence) 1 56 ESRLgGTGAF 12.000 2 20 GSGVrIVVEY 10.800 3 1MSGEpGQTSV 4.000 4 105 ITNSrPPCVI 3.000 5 37 ATYLeLASAV 3.000 6 96ASNGeTLEKI 2.640 7 44 SAVKeQYPGI 2.000 8 8 TSVApPPEEV 2.000 9 74VFSKlENGGF 0.800 10 61 GTGAfEIEIN 0.480 11 26 VVEYcEPCGF 0.400 12 36EATYlELASA 0.360 13 95 RASNgETLEK 0.330 14 63 GAFEiEINGQ 0.264 15 83FPYEkDLIEA 0.240 16 29 YCEPcGFEAT 0.240 17 33 CGFEaTYLEL 0.220 18 43ASAVkEQYPG 0.220 19 75 FSKLeNGGFP 0.200 20 7 QTSVaPPPEE 0.200 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected Cw_0301 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 65 FEIEINGQL 30.000 2 81 GGFPYEKDL18.000 3 70 NGQLVFSKL 12.000 4 57 SRLGGTGAF 10.000 5 34 GFEATYLEL 10.0006 94 RRASNGETL 5.760 7 27 VEYCEPCGF 5.000 8 67 IEINGQLVF 5.000 9 107NSRPPCVIL 2.000 10 51 PGIEIESRL 1.800 11 15 EEVEPGSGV 1.800 12 38TYLELASAV 1.800 13 21 SGVRIVVEY 1.500 14 25 IVVEYCEPC 1.500 15 88DLIEAIRRA 1.500 16 37 ATYLELASA 1.000 17 45 AVKEQYPGI 0.750 18 97SNGETLEKI 0.750 19 106 TNSRPPCVI 0.750 20 29 YCEPCGFEA 0.500 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected Cw_0301 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 44 SAVKeQYPGI 50.000 2 33CGFEaTYLEL 45.000 3 69 INGQlVFSKL 12.000 4 81 GGFPyEKDLI 3.750 5 106TNSRpPCVIL 3.000 6 29 YCEPcGFEAT 2.500 7 16 EVEPgSGVRI 2.500 8 65FEIEiNGQLV 2.160 9 31 EPCGfEATYL 2.000 10 64 AFEIeINGQL 2.000 11 53IEIEsRLGGT 1.500 12 83 FPYEkDLIEA 1.500 13 76 SKLEnGGFPY 1.500 14 21SGVRiVVEYC 1.500 15 37 ATYLeLASAV 1.200 16 80 NGGFpYEKDL 1.200 17 50YPGIeIESRL 1.200 18 93 IRRAsNGETL 1.152 19 23 VRIVvEYCEP 1.000 20 8TSVApPPEEV 1.000 User Parameters and Scoring Information method selectedto limit number of results explicit number number of results requested 20 HLA molecule type selected Cw_0401 length selected for subsequencesto be   9 scored echoing mode selected for input sequence Y echoingformat numbered lines length of user's input peptide sequence 115 numberof subsequence scores calculated 107 number of top-scoring subsequencesreported  20 back in scoring output table Scoring Results Score(Estimate of Half Time of Disassociation of a Molecule Start SubsequenceResidue Containing Rank Position Listing This Subsequence) 1 34GFEATYLEL 240.000 2 38 TYLELASAV 30.000 3 82 GFPYEKDLI 25.000 4 18EPGSGVRIV 20.000 5 31 EPCGFEATY 12.000 6 81 GGFPYEKDL 4.800 7 107NSRPPCVIL 4.800 8 70 NGQLVFSKL 4.400 9 75 FSKLENGGF 2.000 10 97SNGETLEKI 1.584 11 64 AFEIEINGQ 1.000 12 84 PYEKDLIEA 1.000 13 49QYPGIEIES 1.000 14 21 SGVRIVVEY 1.000 15 2 SGEPGQTSV 0.660 16 28EYCEPCGFE 0.600 17 45 AVKEQYPGI 0.600 18 9 SVAPPPEEV 0.600 19 105ITNSRPPCV 0.550 20 77 KLENCGFPY 0.500 User Parameters and ScoringInformation method selected to limit number of results explicit numbernumber of results requested  20 HLA molecule type selected Cw_0401length selected for subsequences to be  10 scored echoing mode selectedfor input sequence Y echoing format numbered lines length of user'sinput peptide sequence 115 number of subsequence scores calculated 106number of top-scoring subsequences reported  20 back in scoring outputtable Scoring Results Score (Estimate of Half Time of Disassociation ofa Molecule Start Subsequence Residue Containing Rank Position ListingThis Subsequence) 1 64 AFEIeINGQL 200.000 2 74 VFSKlENGGF 100.000 3 50YPGIeIESRL 80.000 4 31 EPCGfEATYL 80.000 5 18 EPGSgVRIVV 10.000 6 34GFEAtYLELA 10.000 7 28 EYCEpCGFEA 6.000 8 33 CGFEaTYLEL 5.760 9 84PYEKdLIEAI 5.000 10 83 FPYEkDLIEA 4.800 11 69 INGQlVFSKL 4.400 12 80NGGFpYEKDL 4.000 13 106 TNSRpPCVIL 4.000 14 56 ESRLgGTCAF 2.000 15 66EIEInGQLVF 2.000 16 26 VVEYcEPCGP 2.000 17 96 ASNGeTLEKI 1.320 18 49QYPGiEIESR 1.100 19 20 GSGVrIVVEY 1.000 20 38 TYLElASAVK 0.792 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected Cw_0602 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 85 YEKDLIEAI 6.600 2 65 FEIEINGQL6.600 3 21 SGVRIVVEY 6.000 4 31 EPCGFEATY 3.300 5 61 GTGAFEIEI 3.000 638 TYLELASAV 3.000 7 18 EPGSGVRIV 2.420 8 81 GGFPYEKDL 2.200 9 94RRASNGETL 2.200 10 97 SNGETLEKI 2.000 11 70 NGQLVFSKL 2.000 12 34GFEATYLEL 2.000 13 107 NSRPPCVIL 2.000 14 105 ITNSRPPCV 1.100 15 47KEQYPGIEI 1.100 16 66 EIEINGQLV 1.100 17 42 LASAVKEQY 1.100 18 77KLENGGFPY 1.100 19 15 EEVEPGSGV 1.000 20 45 AVKEQYPGI 1.000 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected Cw_0702 length selected for subsequences to be   9 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 107 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 31 EPCGFEATY 24.000 2 21 SGVRIVVEY19.200 3 42 LASAVKEQY 8.800 4 77 KLENGGFPY 4.000 5 49 QYPGIEIES 2.880 657 SRLGGTGAF 2.400 7 18 EPGSGVRIV 2.400 8 94 RRASNGETL 2.400 9 85YEKDLIEAI 1.478 10 34 GFEATYLEL 1.440 11 38 TYLELASAV 1.440 12 70NGQLVFSKL 1.440 13 65 FEIEINGQL 1.200 14 81 GGFPYEKDL 1.008 15 67IEINGQLVF 1.000 16 97 SNGETLEKI 0.960 17 61 GTGAFEIEI 0.960 18 107NSRPPCVIL 0.840 19 22 GVRIVVEYC 0.800 20 35 FEATYLELA 0.800 UserParameters and Scoring Information method selected to limit number ofresults explicit number number of results requested  20 HLA moleculetype selected Cw_0702 length selected for subsequences to be  10 scoredechoing mode selected for input sequence Y echoing format numbered lineslength of user's input peptide sequence 115 number of subsequence scorescalculated 106 number of top-scoring subsequences reported  20 back inscoring output table Scoring Results Score (Estimate of Half Time ofDisassociation of a Molecule Start Subsequence Residue Containing RankPosition Listing This Subsequence) 1 20 GSGVrIVVEY 38.400 2 30CEPCgFEATY 16.000 3 41 ELASaVKEQY 16.000 4 50 YPGIeIESRL 7.920 5 76SKLEnGGFPY 4.000 6 69 INGQlVFSKL 2.880 7 18 EPGSgVRIVV 2.400 8 33CGFEaTYLEL 1.440 9 80 NGGFpYEKDL 1.440 10 56 ESRLgGTGAF 1.200 11 93IRRAsNGETL 1.200 12 64 AFEIeINGQL 1.200 13 66 EIEInGQLVF 1.000 14 35FEATyLELAS 0.960 15 87 KDLIeAIRRA 0.800 16 97 SNGEtLEKIT 0.800 17 17VEPGsGVRIV 0.800 18 21 SGVRiVVEYC 0.800 19 28 EYCEpCGFEA 0.720 20 48EQYPgIEIES 0.672 Echoed User Peptide Sequence (length = 115 residues)

TABLE 3 File Name: C35 Prediction Parameters: Quantitative Threshold (

): 3 Inhibitor Threshold (log of fold change): −1 Inhibitor Residues(number): 1 0--------30---- DRB1*0101:

VVEYCEPC

DRB1*0301:

VVEYCEPC

DRB1*0401:

VVEYC

DRB1*0701:

DRB1*0801:

DRB1*1101:

DRB1*1501:

DRB5*0101:

RIVVEYCR

(binding frame for

*0101 contains 1 inhibitory residue −100 fold) Quantitative Analysis of‘SGVRIVVEYCEPCGF’ Threshold (

): 10  09  08  07  06  05  04  03  02  01 DRB1*0101XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0102XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0301XXXXXXXXXXXXXXXXXX.................... DRB1*0401XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX DRB1*0402XX.................................... DRB1*0404XXXXXXXXXXXXXXXXXX.................... DRB1*0405XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0410XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*0421XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0701XXXXXXXXXX............................ DRB1*0801XXXXXXXXXXXXXXXXXXXXXX................ DRB1*0802XXXXXX................................ DRB1*0804XXXXXXXXXXXXXX........................ DRB1*0806XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*1101XXXXXXXXXX............................ DRB1*1104XXXXXXXXXX............................ DRB1*1106XXXXXXXXXX............................ DRB1*1107XXXXXXXXXXXXXXXXXX.................... DRB1*1305XXXXXXXXXXXXXXXXXX.................... DRB1*1307XXXXXXXXXX............................ DRB1*1311XXXXXXXXXX............................ DRB1*1321XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*1501XXXXXXXXXX............................ DRB1*1502XXXXXXXXXX............................ DRB5*0101XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ File Name: C35 PredictionParameters: Quantitative Threshold (

): 3 Inhibitor Threshold (log of fold change): −1 Inhibitor Residues(number): 1 ---60--------70---- DRB1*0101: SRLGGTGAFEIEINGQLVFDRB1*0301: SRLGGTGAFEIEINGQLVF DRB1*0401: SRLGGTGA

VF DRB1*0701: SRLGGTGAFEIEINGQLVF DRB1*0801: SRLGGTGAFEIEINGQLVFDRB1*1101: SRLGGTGAFEIEINGQLVF DRB1*1501: SRLGGTGAFEIEINGQLVF DRB5*0101:SRLGGTGAFEIEINGQLVF (binding frame for *0401 contains 2 inhibitoryresidues −10 fold each) Quantitative Analysis of ‘SRLGGTGAFEIEINGQLVF’Threshold (

): 10  09  08  07  06  05  04  03  02  01 DRB1*0101XXXXXXXXXXXXXXXXXXXXXX................ DRB1*0102XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0301XXXXXXXXXXXXXXXXXXXXXX................ DRB1*0401XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0402XXXXXXXXXX............................ DRB1*0404...................................... DRB1*0405XXXXXXXXXX............................ DRB1*0410XX.................................... DRB1*0421XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0701XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX DRB1*0801...................................... DRB1*0802...................................... DRB1*0804XXXXXX................................ DRB1*0806XXXXXX................................ DRB1*1101XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1104XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1106XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1107XX.................................... DRB1*1305XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*1307XX.................................... DRB1*1311XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1321XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1501XXXXXXXXXXXXXXXXXX.................... DRB1*1502XXXXXXXXXXXXXXXXXX.................... DRB5*0101XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ File Name: C35 PredictionParameters: Quantitative Threshold (

): 3 Inhibitor Threshold (log of fold change): −1 Inhibitor Residues(number): 1 -------70--------80-- DRB1*0101: GAFEIEINGQLVFSKLENGGFDRB1*0301: GAFEIEINGQLVFSKLENGGF DRB1*0401: GA

VFSKLENGGF DRB1*0701: GAFEIEINGQLVFSKLENGGF DRB1*0801:GAFEIEINGQLVFSKLENGGF DRB1*1101: GAFEIEINGQLVFSKLENGGF DRB1*1501:GAFEIEINGQLVFSKLENGGF DRB5*0101: GAFEIEINGQLVFSKLENGGF (binding framefor *0401 contains 2 inhibitory residues −10 fold each) QuantitativeAnalysis of ‘GAFEIEINGQLVFSKLENGGF’ Threshold (

): 10  09  08  07  06  05  04  03  02  01 DRB1*0101XXXXXXXXXXXXXXXXXX.................... DRB1*0102XXXXXXXXXXXXXX........................ DRB1*0301XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0401XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0402XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0404XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0405XXXXXXXXXX............................ DRB1*0410XXXXXX................................ DRB1*0421XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*0701XXXXXXXXXXXXXXXXXX.................... DRB1*0801...................................... DRB1*0802...................................... DRB1*0804XXXXXX................................ DRB1*0806XXXXXX................................ DRB1*1101XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1104XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1106XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1107XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX.... DRB1*1305XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*1307XXXXXXXXXXXXXXXXXX.................... DRB1*1311XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*1321XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1501XXXXXXXXXXXXXX........................ DRB1*1502XXXXXXXXXXXXXX........................ DRB5*0101XXXXXXXXXXXXXXXXXXXXXXXXXX............ File Name: C35 PredictionParameters: Quantitative Threshold (

): 5 Inhibitor Threshold (log of fold change): −1 Inhibitor Residues(number): 1 -------90--------100- DRB1*0101: FPYEKDLIEAIRRASNGETLEDRB1*0301: FPYEKDLIEAIRRASNGETLE DRB1*0401: FPYEKDLIEA

LE DRB1*0701: FPYEKDLIEAIRRASNGETLE DRB1*0801: FPYEKDLIEAIRRASNGETLEDRB1*1101: FPYEKDLIEAIRRASNGETLE DRB1*1501: FPYEKDLIEAIRRASNGETLEDRB5*0101: FPYEKDLIEAIRRASNGETLE Quantitative Analysis of‘FPYEKDLIEAIRRASNGETLE’ Threshold (

): 10  09  08  07  06  05  04  03  02  01 DRB1*0101XXXXXXXXXXXXXXXXXX.................... DRB1*0102XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0301XXXXXXXXXXXXXXXXXXXXXX................ DRB1*0401XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0402XXXXXXXXXXXXXXXXXX.................... DRB1*0404XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0405XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0410XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX........ DRB1*0421XXXXXXXXXXXXXXXXXXXXXXXXXX............ DRB1*0701XXXXXXXXXX............................ DRB1*0801XXXXXXXXXXXXXXXXXX.................... DRB1*0802XXXXXXXXXXXXXXXXXX.................... DRB1*0804XXXXXXXXXXXXXXXXXXXXXX................ DRB1*0806XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1101XXXXXXXXXX............................ DRB1*1104XXXXXXXXXXXXXXXXXX.................... DRB1*1106XXXXXXXXXXXXXXXXXX.................... DRB1*1107XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1305XXXXXX................................ DRB1*1307XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1311XXXXXXXXXXXXXXXXXX.................... DRB1*1321XXXXXXXXXXXXXXXXXXXXXX................ DRB1*1501XXXXXXXXXXXXXXXXXXXXXXXXXXXXX......... DRB1*1502XXXXXXXXXXXXXXXXXXXXXXXXXXXXX......... DRB5*0101XXXXXXXXXXXXXX........................ IMPORTANT NOTE:

 was programmed to evaluate

 and

 it was not possible to

 test peptides

 So, whenever the pridicted

 we suggest you should have them

 REPLACING Cys with

indicates data missing or illegible when filed

Altered Peptide Ligands

Identification of immunodominant epitopes of C35 for MHC class Iantigens using specific human T cell lines is a key step toward theirsuccessful use in cancer vaccines. Modified C35 peptide epitomescontaining amino acid substitutions at MHC binding residues have thepotential to be used for enhancement of immune function. Such alteredpeptide ligand, or heteroclitic peptides, can become strong T cellagonists even at 100-fold lower concentrations that the original peptide(Dressel, A. et al., “Autoantigen recognition by human CD8 T Cellclones: enhanced agonist response induced by altered peptide ligand,” J.Immunol. 159:4943-51 (1997). These altered peptide ligand can be of twoforms: those modifications that enhance T cell receptor contact with thepeptide (must be determined experimentally) and those that enhance HLAbinding of the peptide by improving the anchor residues. Table 4specifies modifications that enhance HLA Class I binding by introducingfavorable anchor residues or replacing deleterious residues.

TABLE 4 Modifications that Enhance HLA Class I Binding (Unless otherwiseindicated, examples apply to peptides of 9 amino acids; for 10-mers theamino acid at position 5 is disregarded and the resultant 9-mer isevaluated (http://bimas.dcrt.nih.gov/cgi-bin/ molbio/hla_coefficientviewing_page. The modifications listed below are provided by way ofexample based on current data in existing databases and are not intendedin any way to be an inclusive list of all potential alterations ofpeptides binding all potential HLA molecules, both known and unknown todate.) HLA A*0101 Any altered peptide that has S or T at position 2 Anyaltered peptide that has D or E at position 3 Any altered peptide thathas P at position 4 Any altered peptide that has A, F, I, L, M, P, V, orY at position 7 Any altered peptide that has F, K, R, or Y at anchorposition 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: D, E, F, G, H, K, M, N, P,Q, R, W, Y P3: E, K, R, W P4: K, R P7: D, E, G, R P9: D, E, P HLA A*0201Any altered peptide that has F, I, K, L, M, V, W, or Y at position 1 Anyaltered peptide that has I, L, M, Q, or V at anchor position 2 Anyaltered peptide that has F, L, M, W, or Y at position 3 Any alteredpeptide that has D or E at position 4 Any altered peptide that has F atposition 5 Any altered peptide that has F, I, L, M, V, W or Y atauxiliary anchor position 6 Any altered peptide that has F, or W atposition 7 Any altered peptide that has F, W, or Y at position 8 Anyaltered peptide that has I, L, T or V at anchor position 9 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: D, E, H, P P2: C, F, H, K, N, P, R, S, W, Y P3: D, E, K, RP7: D, E, G, R P8: I, V P9: D, E, F, G, H, K, N, P, Q, R, S, W, YHLA-A*0205 Any altered peptide that has F, I, K, L, M, V, W, or Y atposition 1 Any altered peptide that has E, I, L, M, Q, or V at anchorposition 2 Any altered peptide that has F, L, M, W, or Y at position 3Any altered peptide that has D or E at position 4 Any altered peptidethat has F, Y at position 5 Any altered peptide that has F, I, L, M, V,W or Y at auxiliary anchor position 6 Any altered peptide that has F, orW at position 7 Any altered peptide that has F, W, or Y at position 8Any altered peptide that has I, L, T or V at anchor position 9 Anyaltered peptide where deleterious residues at the following positionsare replaced: P1: D, E, P P2: C, D, F, G, H, K, N, P, R, S, W, Y P3: D,E, K, R P7: D, E, R P9: D, E, F, G, H, K, N, P, Q, R, S, W, Y HLA-A*03Any altered peptide that has G or K at position 1 Any altered peptidethat has I, L, M, Q, T or V at anchor position 2 Any altered peptidethat has F, I, L, M, V, W, or Y at position 3 Any altered peptide thathas E, G or P at position 4 Any altered peptide that has F, I, P, V, W,Y at position 5 Any altered peptide that has F, I, L, M, or V atposition 6 Any altered peptide that has F, I, L, M, W, or Y at position7 Any altered peptide that has F, I, K, L, Q or Y at anchor position 9Any altered peptide where deleterious residues at the followingpositions are replaced: P1: D, E, P P2: D, E, F, G, H, K, N, R, S, W, YP7: G, K, R P9: D, E, G, H, N, P, Q, S, T HLA-A*1101 Any altered peptidethat has G, K or R at position 1 Any altered peptide that has I, L, M,Q, T, V, Y at anchor position 2 Any altered peptide that has F, I, L, M,V, W, Y at position 3 Any altered peptide that has F, I, L, M, W or Y atposition 7 Any altered peptide that has K or R at anchor position 9 Anyaltered peptide where deleterious residues at the following positionsare replaced: P1: D, E, P P2: D, E, G, H, K, N, R, S, W P7: K, R P9: C,D, E, G, N, P, Q, S, T HLA-A24 Any altered peptide that has K or R atposition 1 Any altered peptide that has F or Y at anchor position 2 Anyaltered peptide that has E, I, L, M, N, P, Q, or V at position 3 Anyaltered peptide that has D, E, or P at position 4 Any altered peptidethat has I, L, or V at position 5 Any altered peptide that has F atposition 6 Any altered peptide that has N or Q at position 7 Any alteredpeptide that has E or K at position 8 Any altered peptide that has F, I,L, or M at anchor position 9 Any altered peptide where deleteriousresidues at the following positions are replaced: P1: P P2: D, E, H, K,R P9: D, E, G, H, K, P, Q, R HLA-A*3101 Any altered peptide that has Kor R at position 1 Any altered peptide that has F, I, L, M, Q, T, V, orY at anchor position 2 Any altered peptide that has F, I, L, M, V W, orY at position 3 Any altered peptide that has F, I, L, M, or V atposition 6 Any altered peptide that has F, I, L, M, W, or Y at position7 Any altered peptide that has K or R at anchor position 9 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: D, E, P P2: D, E, G, H, K, N, R, S P7: K, R P9: C, G, N,P, Q, S, T HLA-A*3302 Any altered peptide that has D or E at position 1Any altered peptide that has I, L, M, S, V or Y at anchor position 2 Anyaltered peptide that has R at anchor position 9 Any altered peptidewhere deleterious residues at the following positions are replaced: P1:K, P, R P2: D, E, K, R P9: D, E, F, G, N, P, W, Y HLA-B7 Any alteredpeptide that has A at position 1 Any altered peptide that has A, P or Vat anchor position 2 Any altered peptide that has M or R at position 3Any altered peptide that has P at position 5 Any altered peptide thathas R at position 6 Any altered peptide that has I, L, M or V at anchorposition 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: D, E, F, H, K, R, W, Y P3:D, E P9: D, E, F, G, H, K, N, P, Q, R, S, W, Y HLA-B8 Any alteredpeptide that has D or E at position 1 Any altered peptide that has A, C,L, or P at anchor position 2 Any altered peptide that has K or R atposition 3 Any altered peptide that has D or E at position 4 Any alteredpeptide that has K or R at position 5 Any altered peptide that has I, L,M, or V at anchor position 9 Any altered peptide where deleteriousresidues at the following positions are replaced: P1: K, P, R P2: D, E,F, G, H, K, Q, R, W, or Y P3: D, E P5: D, E P9: D, E, F, G, H, K, N, P,Q, R, S, W, Y HLA-B8 (8-mer peptides) Any altered peptide that has D orE at position 1 Any altered peptide that has A, C, L, or P at anchorposition 2 Any altered peptide that has K or R at position 3 Any alteredpeptide that has D or E at position 4 Any altered peptide that has K orR at position 5 Any altered peptide that has I, L, M, or V at anchorposition 8 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: K, P, R P2: D, E, F, G, H, K, Q,R, W, or Y P3: D, E P5: D, E P8: D, E, F, G, H, K, N, P, Q, R, S, W, YHLA-B14 Any altered peptide that has D or E at position 1 Any alteredpeptide that has K or R at anchor position 2 Any altered peptide thathas F, I, L, M, P, V, W, Y at position 3 Any altered peptide that has Hor R at position 5 Any altered peptide that has I, L, M, R, or V atposition 6 Any altered peptide that has T at position 7 Any alteredpeptide that has I, L, M, or V at anchor position 9 Any altered peptidewhere deleterious residues at the following positions are replaced: P1:P P2: D, E, F, W, or Y P3: E, R P5: E, W, Y P9: D, E, G, H, K, N, P, Q,R HLA-B*2702 Any altered peptide that has K or R at position 1 Anyaltered peptide that has E, L, M, N, Q or R at anchor position 2 Anyaltered peptide that has F, W, or Y at position 3 Any altered peptidethat has F, I, L, W or Y at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: D, E,P P2: D, F, G, H, K, W, or Y P7: K P9: D, E, G, K, N, P, Q, R, SHLA-B27*05 (8-mer peptides) Any altered peptide that has K or R atposition 1 Any altered peptide that has E, L, M, N, Q or R at anchorposition 2 Any altered peptide that has F, W, or Y at position 3 Anyaltered peptide that has F, I, K, L, M, R, V or Y at anchor position 8Any altered peptide where deleterious residues at the followingpositions are replaced: P1: D, E, P P2: D, F, G, H, K, W, or Y P7: K P9:D, E, G, K, N, P, Q, R, S HLA-B*3501 (8-mer peptides) Any alteredpeptide that has K or R at position 1 Any altered peptide that has A, P,or S at anchor position 2 Any altered peptide that has K or R atposition 3 Any altered peptide that has D or E at position 4 Any alteredpeptide that has D or E at position 5 Any altered peptide that has F, I,L, M, V, W or Y at anchor position 8 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P2:D, E, F, H, K, R, W, Y P3: D, E P8: D, E, F, G, H, K, P, Q, R HLA-B*3701Any altered peptide that has D or E at anchor position 2 Any alteredpeptide that has I or V at position 5 Any altered peptide that has F, L,or M at position 8 Any altered peptide that has F, I, L, M, V or Y atanchor position 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P9: D, E, G, H, K, P, Q, RHLA-B*3801 Any altered peptide that has F, H, P, W or Y at anchorposition 2 Any altered peptide that has D or E at position 3 Any alteredpeptide that has D, E, or G at position 4 Any altered peptide that hasA, I, L, M, or V at position 5 Any altered peptide that has K or Y atposition 8 Any altered peptide that has F, I, L, M, or V at anchorposition 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: D, E, K, R P3: K, R P9: D,E, G, H, K, P, Q, R HLA-B*3901 (8-mer peptides) Any altered peptide thathas H or R at anchor position 2 Any altered peptide that has D, E, F, I,L, M, V, W, or W at position 3 Any altered peptide that has D or E atposition 4 Any altered peptide that has I, L, M, or V at position 6 Anyaltered peptide that has I, L, M or V at anchor position 8 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: P P2: D, E P3: K, R P6: D, E, K, R P8: D, E, G, H, K, P,Q, R HLA-B*3902 Any altered peptide that has K or Q at anchor position 2Any altered peptide that has F, I, L, M, V, W, or Y at position 5 Anyaltered peptide that has F, L, or M at anchor position 9 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: P P2: D, E P3: K, R P9: D, E, G, H, K, P, Q, R HLA-B40 Anyaltered peptide that has A or G at position 1 Any altered peptide thathas D or E at anchor position 2 Any altered peptide that has A, F, I, L,M, V, W, or Y at position 3 Any altered peptide that has P at position 4Any altered peptide that has P at position 5 Any altered peptide thathas A, L, M, or W at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P2:F, H, I, K, L, M, Q, R, V, W, or Y P3: D, E, K, R P9: D, E, G, H, K, N,P, Q, R HLA-B44*03 Any altered peptide that has A, D, or S at position 1Any altered peptide that has D or E at anchor position 2 Any alteredpeptide that has A, I, L, M, or V at position 3 Any altered peptide thathas F, I, or P at position 4 Any altered peptide that has A, K, or V atposition 5 Any altered peptide that has A, L, T, or V at position 6 Anyaltered peptide that has F, K, or T at position 7 Any altered peptidethat has K at position 8 Any altered peptide that has F, W or Y atanchor position 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: F, H, I, K, L, M, Q, R, V,W, Y P9: D, E, G, H, K, N, P, Q, R HLA-B*5101 (8-mer peptides) Anyaltered peptide that has D, E, F, I, L, M, V, or Y at position 1 Anyaltered peptide that has A, G or P at anchor position 2 Any alteredpeptide that has F, W or Y at position 3 Any altered peptide that has D,E, G, I, K, or V at position 4 Any altered peptide that has A, G, I, S,T, or V at position 5 Any altered peptide that has I, K, L, N, or Q atposition 6 Any altered peptide that has D, K, Q, or R at position 7 Anyaltered peptide that has I, L, M, or V at anchor position 8 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: K, P, R P2: D, E, H, K P8: D, E, F, G, H, K, N, P, Q, R,S, W, Y HLA-B*5102 Any altered peptide that has F or Y at position 1 Anyaltered peptide that has A, G, or P at anchor position 2 Any alteredpeptide that has F, I, L, V, W, or Y at position 3 Any altered peptidethat has E, G, H, K, L, N, Q, R, or T at position 4 Any altered peptidethat has G, N, Q, T, or V at position 5 Any altered peptide that has I,N, Q, or T at position 6 Any altered peptide that has E, K, Q, or R atposition 7 Any altered peptide that has K, R, T, or Y at position 8 Anyaltered peptide that has I, L, M, or V at anchor position 9 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: P P2: D, E, H, K, R P3: D, E, K, R P9: D, E, F, G, H, K,N, P, Q, R, S, W, Y HLA-B*5102 (8-mer peptides) Any altered peptide thathas F or Y at position 1 Any altered peptide that has A, G, or P atanchor position 2 Any altered peptide that has F, I, L, V, W, or Y atposition 3 Any altered peptide that has E, G, H, K, L, V, W, or Y atposition 4 Any altered peptide that has G, N, Q, T, V at position 5 Anyaltered peptide that has I, N, or Q at position 6 Any altered peptidethat has Q, or R at position 7 Any altered peptide that has I, L, M, orV at position 8 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: D, E, H, K, R P3: D, E, K, RP8: D, E, F, G, H, K, N, P, Q, R, S, W, Y HLA-B*5103 Any altered peptidethat has D, T, or V at position 1 Any altered peptide that has A, G, orP at anchor position 2 Any altered peptide that has D, F, L, or Y atposition 3 Any altered peptide that has E, G, L, N, Q, R, T, or V atposition 4 Any altered peptide that has A, G, M, N, Q, R, K or V atposition 5 Any altered peptide that has I, K, or T at position 6 Anyaltered peptide that has M or V at position 7 Any altered peptide thathas I, L, M, or V at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P2:D, E, H, K, R P9: D, E, F, G, H, K, N, P, Q, R, S, W, Y HLA-B*5201(8-mer peptides) Any altered peptide that has I, L, M, or V at position1 Any altered peptide that has G, P, or Q at anchor position 2 Anyaltered peptide that has D, F, I, L, P, W, or Y at position 3 Anyaltered peptide that has A, E, I, K, L, P, or V at position 4 Anyaltered peptide that has A, F, G, I, L, M, T or V at position 5 Anyaltered peptide that has K, L, N, S or T at position 6 Any alteredpeptide that has E, K, Q, or Y at position 7 Any altered peptide thathas F, I, L, M, or V at anchor position 8 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P2:H, K, R P3: R P8: D, E, G, H, K, N, P, Q, R, S HLA-B*5801 Any alteredpeptide that has I, K, or R at position 1 Any altered peptide that hasA, S, or T at anchor position 2 Any altered peptide that has D atposition 3 Any altered peptide that has E, K, or P at position 4 Anyaltered peptide that has F, I, L, M, or V at position 5 Any alteredpeptide that has F, I, L, or V at position 6 Any altered peptide thathas L, M, N, or Y at position 7 Any altered peptide that has K, N, R, orT at position 8 Any altered peptide that has F, W, or Y at anchorposition 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: D, E, P P2: D, E, F, H, I, K, L,M, N, Q, R, V, W, Y P9: D, E, G, H, K, N, P, Q, R, S HLA-B*60 Anyaltered peptide that has D or E at anchor position 2 Any altered peptidethat has A, I, L, M, S, or V at position 3 Any altered peptide that hasL, I, or V at position 5 Any altered peptide that has I, L, M, V, or Yat position 7 Any altered peptide that has K, Q, or R at position 8 Anyaltered peptide that has I, L, M, or V at anchor position 9 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: P P2: F, H, I, K, L, M, Q, R, V, W, Y P9: D, E, F, G, H,K, N, P, Q, R, S, W, Y HLA-B*61 Any altered peptide that has G or R atposition 1 Any altered peptide that has D or E at anchor position 2 Anyaltered peptide that has A, F, I, L, M, T, V, W, or Y at position 3 Anyaltered peptide that has I at position 6 Any altered peptide that has Yat position 7 Any altered peptide that has A, I, L, M, or V at anchorposition 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: F, H, I, K, L, M, Q, R, V,W, Y P9: D, E, F, G, H, K, N, P, Q, R, S, W, Y HLA-B*61 (8-mer peptides)Any altered peptide that has G or R at position 1 Any altered peptidethat has D or E at anchor position 2 Any altered peptide that has A, F,I, L, M, T, V, W, or Y at position 3 Any altered peptide that has I atposition 6 Any altered peptide that has Y at position 7 Any alteredpeptide that has A, I, L, M, or V at anchor position 8 Any alteredpeptide where deleterious residues at the following positions arereplaced: P1: P P2: F, H, I, K, L, M, Q, R, V, W, Y P8: D, E, F, G, H,K, N, P, Q, R, S, W, Y HLA-B*62 Any altered peptide that has I atposition 1 Any altered peptide that has I, L, Q at anchor position 2 Anyaltered peptide that has G, K, R at position 3 Any altered peptide thathas D, E, G, or P at position 4 Any altered peptide that has F, G, I, L,or V at position 5 Any altered peptide that has I, L, T, V at position 6Any altered peptide that has T, V, or Y at position 7 Any alteredpeptide that has F, W, Y at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P2:D, E, F, H, K, N, R, S, W, Y P3: D, E P6: D, E, K, R P9: D, E, G, H, K,N, P, Q, R, S HLA-Cw0301 Any altered peptide that has A or R at anchorposition 2 Any altered peptide that has F, I, L, M, V, or Y at position3 Any altered peptide that has E, P, or R at position 4 Any alteredpeptide that has N at position 5 Any altered peptide that has F, M, or Yat position 6 Any altered peptide that has K, M, R, or S at position 7Any altered peptide that has T at position 8 Any altered peptide thathas F, I, L, M at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P3:D, K, R P6: D, E, K, R P9: D, E, G, H, K, N, P, Q, R, S, HLA-Cw0401 Anyaltered peptide that has F, P, W, or Y at anchor position 2 Any alteredpeptide that has D, or H at position 3 Any altered peptide that has D orE at position 4 Any altered peptide that has A, H, M, R, or T atposition 5 Any altered peptide that has I, L, M, or V at position 6 Anyaltered peptide that has A at position 7 Any altered peptide that has H,K, or S at position 8 Any altered peptide that has F, I, L, M, V or Y atanchor position 9 Any altered peptide where deleterious residues at thefollowing positions are replaced: P1: P P2: D, E, H, K, R P9: D, E, G,H, K, N, P, Q, R, S HLA-Cw0602 Any altered peptide that has F, I, K, orY at position 1 Any altered peptide that has A, P, Q, or R at anchorposition 2 Any altered peptide that has F, I, K, L, or M at position 5Any altered peptide that has I, L, or V at position 6 Any alteredpeptide that has K, N, Q, or R at position 7 Any altered peptide thathas I, L, M, V, or Y at anchor position 9 Any altered peptide wheredeleterious residues at the following positions are replaced: P1: P P9:D, E, G, H, K, N, P, Q, R, S

Examples of predicted human Class I MHC binding peptides from the C35 aasequence and how they might be changed to improve binding: Start Score(estimated Rank Position Subsequence halftime of dissociation) SEQ IDNO. HLA-A*0101 1  77 KLENGGRPY   225.000 2  16 EVEPGSGVR    90.000 3  29YCEPCGFEA    45.000 4  39 YLELASAVK    36.000 5   2 SGEPGQTSV     2.250G is deleterious at P2 example of STEPGQTSV    22.50 G replaced with T@ P2 SEQ ID NO: 85 improved peptide example of STEPGQISY  5625.00 V atP9 replaced SEQ ID NO: 86 improved peptide with Y, P7 enhancedHLA-A*0101 (10-mer peptides) 1  66 EIEINGQLVF    45.000 2  16 EVEPGSGVRI   18.000 3  29 YCEPCGFEAT     9.000 4  26 VVEYCEPCGF     9.000 5  52GIEIESRLGG     2.250 example of GTEPSRLGY  1125.000 replace I with T@ P2 SEQ ID NO: 87 improved peptide replace G with V @ P9 P5 enhancedwith P HLA-A*0201 (9-mer peptides) 1   9 SVAPPPEEV     2.982 2 104KITNSRPPC     2.391 3 105 ITNSRPPCV     1.642 4  25 IVVEYCEPC     1.4855  65 FEIEINGQL     1.018 example of FLIEINWYL 16619.000 SEQ ID NO: 88improved peptide HLA-A*0201 (10-mer peptides) 1  58 RLGGTGAFEI    60.5102 104 KITNSRPPCV    33.472 3  65 FEIEINGQLV    25.506 4  83 FPYEKDLIEA    4.502 P is deleterious at P2 example of FLYEKDLIEA   689.606 replaceP with L @ P2 SEQ ID NO: 89 improved peptide example of FLYEKDLIEV 9654.485 replace A with V @ P9 SEQ ID NO: 90 improved peptide 5  33CGFEATYLEL     3.173 HLA-A*0205 1  65 FEIEINGQL     8.820 2  25IVVEYCEPC     3.060 3   9 SVAPPPEEV     2.000 4 104 KITNSRPPC     1.5005  81 GGFPYEKDL     1.260 G is deleterious at P2 example of GVFPYEKDL   50.400 replace G with V @ P2 SEQ ID NO: 91 improved peptideHLA-A*0205 (10-mer peptides) 1  33 CGEFATYLEL     6.300 G is deleteriousat P2 example of CVEFATYLEL    11.200 replace G with V @ P2 SEQ ID NO:92 improved peptide 2 104 KITNSRPPCV     6.000 3  65 FEIEINGQLV    2.520 4  53 IEIESRLGGT     1.428 5  83 FPYEKDLIEA     1.350 P isdeleterious at P2 example of FVYEKDLIEA    54.000 replace P with V @ P2SEQ ID NO: 93 improved peptide HLA-A24 1  34 GFEATYLEL    33.000 2  49QYPGIEIES    11.550 example of QYPGIEIEL   462.000 enhance P9 SEQ ID NO:94 improved peptide 3  70 NGQLZFSKL    11.088 4  38 TYLELASAV    10.8005  82 GFPYEKDLI     7.500 HLA-A24 (10-mer peptides) 1  64 AFEIEINGQL   42.000 2  74 VFSKLENGGF    10.000 3  84 PYEKDLIEAI     9.000 4  69INGQLVFSKL     7.392 example of IYGQLVFSKL   369.6 enhance P2 SEQ ID NO:95 improved peptide 5  28 EYCEPCGFEA     6.600 HLA-A3 1  77 KLENGGFPY   36.000 example of KLENGGFPK   180.000 enhance P9 SEQ ID NO: 96improved peptide 2  39 YLELASAVK    20.000 3 101 TLEKITNSR     6.000 4 61 GTGAFEIEI     0.540 5  69 INGQLVFSK     0.360 N is deleterious @ P2example of ILGQLVFSK   180.000 replace N with L @ P2 SEQ ID NO: 97improved peptide HLA-A3 (10-mer peptides) 1  68 EINGQLVFSK     8.100 2 58 RLGGTGAFEI     2.700 3  41 ELASAVKEQY     1.800 4  78 LENGGFPYEK    0.810 E is deleterious @ P2 example of LLNGGFPYEK   270.000 replaceE with L @ P2 SEQ ID NO: 98 improved peptide 5  95 RASNGETLEK     0.400HLA-A*1101 1  39 YLELASAVK     0.400 2  69 INGQLVFSK     0.120 N isdeleterious @ P2 example of IVGQLVFSK     6.000 replace N with V @ P2SEQ ID NO: 99 improved peptide 3  16 EVEPGSGVR     0.120 4 101 TLEKITNSR    0.080 5  61 GTGAFEIEI     0.060 HLA-A*1101 (10-mer peptides) 1  95RASNGETLEK     1.200 2  38 TYLELASAVK     0.600 3  68 EINGGLVFSK    0.360 4  78 LENGGFPYEK     0.120 E is deleterious @ P2 example ofLVNGGFPYEK     4.000 replace E with V @ P2 SEQ ID NO: 100 improvedpeptide 5 100 ETLEKITNSR     0.090 HLA-A*3101 1 101 TLEKITNSR     2.0002  16 EVEPGSGVR     0.600 3  50 YPGIEIESR     0.400 4  87 KDLIEAIRR    0.240 D is deleterious @ P2 example of KILIEAIRR    12.000 replace Dwith I @ P2 SEQ ID NO: 101 improved peptide 5  39 YLELASAVK     0.200HLA-A*3302 1  16 EVEPGSGVR    45.000 2 101 TLEKITNSR     9.000 3  50YPGIEIESR     3.000 4  66 EIEINGQLV     1.500 5  56 ESRLGGTGA     1.500HLA-A*3302 (10-mer peptides) 1  49 QYPGIEIESR    15.000 2 100 ETLEKITNSR    9.000 3  16 EVEPGSGVRI     1.500 4  28 EYCEPCGFEA     1.500 5  68EINGQLVFSK     1.500 HLA-A68.1 1  16 EVEPGSGVR   900.000 2   9 SVAPPPEEV   12.000 3  50 YPGIEIESR    10.000 example of YVGIEIESR   400.000enhance P2 SEQ ID NO: 102 improved peptide 4  96 ASNGETLEK     9.000 5101 TLEKITNSR     5.000 HLA-A68.1 (10-mer peptides) 1 100 ETLEKITNSR  300.000 2  16 EVEPGSGVRI    18.000 3  68 EINGGLVFSK     9.000 4  15EEVEPGSGVR     9.000 E is deleterious @ P2 example of EVVEPGSGR  1200.00replace E with V @ P2 SEQ ID NO: 103 improved peptide 5  95 RASNGETLEK    3.000 HLA-B14 1  94 RRASNGETL    20.000 2  57 SRLGGTGAF     5.000example of SRLGGTGAL   100.000 enhance P9 SEQ ID NO: 104 improvedpeptide 3 100 ETLEKITNS     3.375 4 105 ITNSRPPCV     2.000 5  88DLIEAIRRA     1.350 HLA-B14 (10-mer peptides) 1 103 EKITNSRPPC     6.750example of ERITNSRPPL   900.000 enhance P10 SEQ ID NO: 105 improvedpeptide 2  33 CGFEATYLEL     5.000 3  93 IRRASNGETL     4.000 4  18EPGSGVRIVV     3.000 5  88 DLIEAIRRAS     2.250 HLA-B40 1  65 FEIEINGQL   80.000 2   3 GEPGQTSVA    40.000 3  35 FEATYLELA    40.000 4  15EEVEPGSGV    24.000 example of EEVEPGSGL   120.000 enhance P9 SEQ ID NO:106 improved peptide 5  67 IEINGQLVF    16.000 HLA-B40 (10-mer peptides)1  55 IESRLGGTGA    20.000 2  53 IEIESRLGGT    16.000 example ofIEIESRLGGL    80.000 enhance P10 SEQ ID NO: 107 improved peptide 3  65FEIEINGQLV    16.000 4  67 IEINGQLVFS    16.000 5  99 GETLEKITNS    8.000 HLA-B60 1  65 FEIEFNGQL   387.200 2  17 VEPGSGVRI    17.600example of VEPGSGVRL   352.000 enhance P9 SEQ ID NO: 108 improvedpeptide 3  15 EEVEPGSGV    16.000 4  47 KEQYPGIEI    16.000 5  85YEKDLIEAI     8.800 HLA-B60 (10-mer peptides) 1  65 FEIEINGQLV    16.000example of FEIEINGQLL   320.000 enhance P10 SEQ ID NO: 109 improvedpeptide 2 106 TNSRPPCVIL    16.000 3  53 IEIESRLGGT     8.000 4  33CGFEATYLEL     8.000 5  17 VEPGSGVRIV     8.000 HLA-B61 1  15 EEVEPGSGV   80.000 2  35 FEATYLELA    40.000 example of FEATYLELV   160.000enhance P9 SEQ ID NO: 110 improved peptide 3   3 GEPGQTSVA    22.000 4 65 FEIENGGQL    16.000 5  85 YEKDLIEAI    16.000 HLA-B61 (10-merpeptides) 1  65 FEIEINGQLV    80.000 2  17 VEPGSGVRIV    40.000 3  55IESRLGGTGA    20.000 4  87 KDLIEAIRRA    10.000 example of KELIEAIRRV  160.000 enhance P2, P10 SEQ ID NO: 111 improved peptide 5  53IEIESRLGGT     8.000 HLA-B62 1  77 KLENGGFPY    24.000 2  21 SGVRIVVEY    4.800 3  75 FSKLENGGF     3.000 4  31 EPCGFEATY     2.640 P isdeleterious @ P2 example of EQCGFEATY   105.6 replace P with Q @ P2 SEQID NO: 112 improved peptide 5  88 DLIEAIRRA     2.200 HLA-B62 (10-merpeptides) 1  41 ELASAVKEQY    40.000 2  58 RLGGTGAFEI     9.600 3  66EIEINGQLVF     7.920 4  56 ESRLGGTGAF     6.000 S is deleterious @ P2example of EQRLGGTGAF   480.000 replace S with Q @ P2 SEQ ID NO: 113improved peptide 5  20 GSGVRIVVEY     4.800 S is deleterious @ P2example of GQGVRIVVEY   384.000 replace S with Q @ P2 SEQ ID NO: 114improved peptide HLA-B7 1 107 NSRPPCVIL    60.000 example of NPRPPCVIL 1200.000 enhance P2 SEQ ID NO: 115 improved peptide 2  45 AVKEQYPGI    6.000 3  22 GVRIVVEYC     5.000 4  70 NGQLVFSKL     4.000 5  81GGFPYEKDL     4.000 HLA-B7 (10-mer peptides) 1  50 YPGIEIESRL    80.0002  31 EPCGFEATYL    80.000 3  18 EPGSGVRIVV     6.000 example ofEPGSGVRIVL   120.000 enhance P10 SEQ ID NO: 116 improved peptide 4 106TNSRPPCVIL     6.000 5  80 NGGFPYEKDL     4.000 HLA-B8 1 107 NSRPPCVIL    4.000 2  45 AVKEQYPGI     1.500 3 105 ITNSRPPCV     0.600 4  56ESRLGGTGA     0.400 5 100 ETLEKITNS     0.300 S is deleterious @ P9example of ETLEKITNL    12.000 replace S with L @ P9 SEQ ID NO: 117improved peptide HLA-B8 (8-mer peptides) 1  83 FPYEKDLI     6.000 2 107NSRPPCVI     1.000 3  91 EAIRRASN     0.800 N is deleterious @ P8example of EAIRRASL    32.000 replace N with L @ P9 SEQ ID NO: 118improved peptide 4  20 GSGVRIVV     0.600 5  18 EPGSGVRI     0.400HLA-B8 (10-mer peptides) 1  50 YPGIEIESRL     0.800 2  93 IRRASNGETL    0.400 example of IA RASNGETL    16.000 replace R with A @ P2 SEQ IDNO: 119 improved peptide 3  31 EPCGFEATYL     0.320 4 104 KITNSRPPCV    0.300 5  18 EPGSGVRIVV     0.240 HLA-B*2702 1  57 SRLGGTGAF  200.000 2  94 RRASNGETL   180.000 example of RRASNGETF   600.000enhance P9 SEQ ID NO: 120 improved peptide 3  93 IRRASNGET    20.000 4 27 VEYCEPCGF    15.000 5  77 KLENGGFPY     9.000 HLA-B*2702 (10-merpeptides) 1  93 IRRASNGETL    60.000 2  94 RRASNGETLE     6.000 3  30CEPCGFEATY     3.000 4  58 RLGGTGAFEI     2.700 5  23 VRIVVEYCEP    2.000 P is deleterious @ P10 example of VRIVVEYCEY   200.000 replaceP with V @ P10 SEQ ID NO: 121 improved peptide HLA-B*2705 1  94RRASNGETL  6000.000 2  57 SRLGGTGAF  1000.000 3  93 IRRASNGET   200.000example of IRRASNGEL  2000.000 enhance P9 SEQ ID NO: 122 improvedpeptide 4  27 VEYCEPCGF    75.000 5  77 KLENGGFPY    45.000 HLA-B*2705(10-mer peptides) 1  93 IRRASNGETL  2000.000 2  94 RRASNGETLE    60.000E is deleterious @ P2 example of RRASNGETLL  6000.000 replace E with L@ P2 SEQ ID NO: 123 improved peptide 3  78 LENGGFPYEK    30.000 4  95RASNGETLEK    30.000 5  58 RLGGTGAFEI    27.000 HLA-B*3501 1  31EPCGFEATY    40.000 2  75 FSKLENGGF    22.500 example of FPKLENGGM  120.000 enhance P2, P9 SEQ ID NO: 124 improved peptide 3 107 NSRPPCVIL   15.000 4  42 LASAVKEQY     6.000 5  18 EPGSGVRIV     4.000 HLA-B*3501(10-mer peptides) 1  31 EPCGFEATYL    30.000 2  50 YPGIEIESRL    20.0003  56 ESRLGGTGAF    15.000 4  20 GSGVRIVVEY    10.000 5  83 FPYEKDLIEA    6.000 example of FPYEKDLIEM   120.000 enhance P10 SEQ ID NO: 125improved peptide HLA-B*3701 1  65 FEIEINGQL    15.000 example ofFDIEINGQL    60.000 enhance P2 SEQ ID NO: 126 improved peptide 2  47KEQYPGIEI    10.000 3  85 YEKDLIEAI    10.000 4  17 VEPGSGVRI    10.0005  35 FEATYLELA     5.000 HLA-B*3701 (10-mer peptides) 1  65 FEIEINGQLV   10.000 example of FDIEINGQLI   200.000 enhance P2, P10 SEQ ID NO: 127improved peptide 2  67 IEINGQLVFS     5.000 3  81 GGFPYEKDLI     5.000 4 87 KDLIEAIRRA     4.000 5  30 CEPCGFEATY     2.000 HLA-B*3801 1  34GFEATYLEL     6.000 example of GHEATYLEL    90.000 enhance P2 SEQ ID NO:128 improved peptide 2  70 NGQLVFSKL     1.560 3  38 TYLELASAV     1.0404  81 GGFPYEKDL     1.000 5  97 SNGETLEKI     0.720 HLA-B*3801 (10-merpeptides) 1  64 AFEIEINGQL     7.800 example of AHEIEINGQL   117.000enhance P2 SEQ ID NO: 129 improved peptide 2  31 EPCGFEATYL     4.800 3 66 EIEINGQLVF     3.000 4  26 VVEYCEPCGF     3.000 5  50 YPGIEIESRL    2.600 HLA-B*3901 1  94 RRASNGETL    15.000 example of RHASNGETL   90.000 enhance P2 SEQ ID NO: 130 improved peptide 2  34 GFEATYLEL    9.000 3  38 TYLELASAV     4.000 4  66 EIEINGQLV     3.000 5   2SGEPGQTSV     3.000 HLA-B*3901 (10-mer peptides) 1  33 CGFEATYLEL   12.000 example of CHFEATYLEL   360.000 enhance P2 SEQ ID NO: 131improved peptide 2  64 AFEIEINGQL     9.000 3  93 IRRASNGETL     4.500 4 46 VKEQYPGIEI     3.000 5  16 EVEPGSGVRI     3.000 HLA-B*3902 1  70NGQLVFSKL     2.400 example of NKQLVFSKL    24.000 enhance P2 SEQ ID NO:132 improved peptide 2  81 GGFPYEKDL     2.400 3  94 RRASNGETL     2.0004  34 GFEATYLEL     2.000 5 107 NSRPPCVIL     0.600 HLA-B*3902 (10-merpeptides) 1  69 INGQLVFSKL     2.400 2  64 AFEIEINGQL     2.400 3  50YPGIEIESRL     2.400 4  80 NGGFPYEKDL     2.400 5 106 TNSRPPCVIL    2.000 HLA-B*4403 1  67 IEINGQLVF   200.000 example of IEINGQLVY  900.000 enhance P9 SEQ ID NO: 133 improved peptide 2  27 VEYCEPCGF   40.000 3  21 SGVRIVVEY    36.000 4  65 FEIEINGQL    20.000 5  35FEATYLELA    12.000 HLA-B*4403 (10-mer peptides) 1  30 CEPCGFEATY  120.000 2  53 IEIESRLGGT    30.000 example of IEIESRLGGY   900.000enhance P10 SEQ ID NO: 134 improved peptide 3  67 IEINGQLVFS    30.000 4 65 FEIEINGQLV    20.000 5  17 VEPGSGVRIV    18.000 HLA-B*5101 1  18EPGSGVRIV   484.000 2  59 LGGTGAFEI   114.400 example of LPGTGAFEI  572.000 enhance P2 SEQ ID NO: 135 improved peptide 3   2 SGEPGQTSV   48.400 4  81 GGFPYEKDL    44.000 5  70 NGQLVFSKL    22.000 HLA-B*5101(10-mer peptides) 1  18 EPGSGVRIVV   440.000 2  44 SAVKEQYPGI   220.000example of SPVKEQYPGI   440.000 enhance P2 SEQ ID NO: 136 improvedpeptide 3  31 EPCGFEATYL   220.000 4  81 GGFPYEKDLI   176.000 5  50YPGIEIESRL   157.300 HLA-B*5102 1  18 EPGSGVRIV   242.000 2  81GGFPYEKDL   110.000 example of GPFPYEKDI  2200.000 enhance P2, P9 SEQ IDNO: 137 improved peptide 3  59 LGGTGAFEI    96.800 4  70 NGQLVFSKL   48.400 5   2 SGEPGQTSV    24.200 HLA-B*5102 (10-mer peptide) 1  44SAVKEQYPGI   726.000 example of SPVKEQYPGI  1452.000 enhance P2 SEQ IDNO: 138 improved peptide 2  50 YPGIEIESRL   400.000 3  81 GGFPYEKDLI  400.000 4  18 EPGSGVRIVV   220.000 5  31 EPCGFEATYL   121.000HLA-B*5103 1  59 LGGTGAFEI    48.400 example of LAFTGAFEI   145.200enhance P2 SEQ ID NO: 139 improved peptide 2   2 SGEPGQTSV    44.000 3 18 EPGSGVRIV    44.000 4  70 NGQLVFSKL     7.260 5  81 GGFPYEKDL    7.200 HLA-B*5103 (10-mer peptide) 1  44 SAVKEQYPGI   110.000 2  81GGFPYEKDLI    52.800 3  18 EPGSGVRIVV    44.000 example of EAGSGVRIVV  110.000 enhance P2 SEQ ID NO: 140 improved peptide 4  60 GGTGAFEIEI   44.000 5  33 CGFEATYLEL     7.920 HLA-B*5201 1  18 WPGSGVRIV   75.000 2  67 LEINGQLVF    22.500 example of LQINGQLVI   450.000enhance P2, P9 SEQ ID NO: 141 improved peptide 3  59 LGGTGAFEI    11.2504  98 NGETLEKIT    11.000 5  19 PGSGVRIVV    10.000 HLA-B*5201 (10-merpeptides) 1  18 EPGSGVRIVV   100.000 2  17 VEPGSGVRIV    45.000 exampleof VQPGSGVRIV   450.000 enhance P2 SEQ ID NO: 142 improved peptide 3  81GGFPYEKDLI    33.000 4 105 ITNSRPPCVI    15.000 5  37 ATYLELASAV   12.000 HLA-B*5801 1  75 FSKLENGGF    40.000 example of FSKLENGGW   80.000 enhance P9 SEQ ID NO: 143 improved peptide 2  42 LASAVKEQY    4.500 3 107 NSRPPCVIL     4.000 4  61 GIGAFEIEI     3.000 5 105ITNSRPPCV     3.000 HLA-B*5801 (10-mer peptides) 1  56 ESRLGGTGAF   12.000 2  20 GSGVRIVVEY    10.800 example of GSGVRIVVEW   144.000enhance P10 SEQ ID NO: 144 improved peptide 3   1 MSGEPGQTSV     4.000 4105 ITNSRPPCVI     3.000 5  37 ATYLELASAV     3.000 HLA-Cw*0301 1  65FEIEINGQL    30.000 2  81 GGFPYEKDL    18.000 3  70 NGQLVFSKL    12.0004  57 SRLGGTGAF    10.000 5  34 GFEATYLEL    10.000 HLA-Cw*0301 (10-merpeptides) 1  44 SAVKEQYPGI    50.000 example of SAVKEQYPGL   100.000enhance P10 SEQ ID NO: 145 improved peptide 2  33 CGFEATYLEL    45.000 3 69 INGQLVFSKL    12.000 4  81 GGFPYEKDLI     3.750 5 106 TNSRPPCVIL    3.000 HLA-Cw*0401 1  34 GFEATYLEL   240.000 2  38 TYLELASAV   30.000 3  82 GFPYEKDLI    25.000 4  18 EPGSGVRIV    20.000 5  31EPCGFEATY    12.000 example of EFCGFEATL   200.000 enhance P2, P9 SEQ IDNO: 146 improved peptide HLA-Cw*0401 (10-mer peptides) 1  64 AFEIEINGQL  200.000 2  74 VFSKLENGGF   100.000 example of VFSKLENGGL   200.000enhance P10 SEQ ID NO: 147 improved peptide 3  50 YPGIEIESRL    80.000 4 31 EPCGFEATYL    80.000 5  18 EPGSGVRIVV    10.000 HLA-Cw*0602 1  85YEKDLIEAI     6.600 2  65 FEIEINGQL     6.600 3  21 SGVRIVVEY     6.0004  31 EPCGFEATY     3.300 5  61 GTGAGEIEI     3.000 HLA-Cw*0702 1  31EPCGFEATY    24.000 2  21 SGVRIVVEY    19.200 3  42 LASAVKEQY     8.8004  77 KLENGGFPY     4.000 5  49 QYPGIEIES     2.880 HLA-Cw*0702 (10-merpeptides) 1  20 GSGVRIVVEY    38.400 2  30 CEPCGFEATY    16.000 3  41ELASAVKEQY    16.000 4  50 YPGIEIESRL     7.920 5  76 SKLENGGFPY    4.000

TABLE 5 Predicted C35 HLA Class I epitopes* HLA Inclusive restritionelement amino acids Sequence A*0201   9-17 SVAPPPEEV A*0201  10-17VAPPPEEV A*0201  16-23 EVEPGSGV A*0201  16-25 EVEPGSGVRI A*0201  36-43EATYLELA A*0201  37-45 ATYLELASA A*0201  37-46 ATYLELASAV A*0201  39-46YLELASAV A*0201  44-53 SAVKEQYPGI A*0201  45-53 AVKEQYPGI A*0201  52-59GIEIESRL A*0201  54-62 EIESRLGGT A*0201  58-67 RLGGTGAFEI A*0201  61-69GTGAFEIEI A*0201  66-73 EIEINGQL A*0201  66-74 EIEINGQLV A*0201  88-96DLIEAIRRA A*0201  89-96 LIEAIRRA A*0201  92-101 AIRRASNGET A*0201 95-102 RASNGETL A*0201 104-113 KITNSRPPCV A*0201 105-113 ITNSRPPCVA*0201 105-114 ITNSRPPCVI A*3101  16-24 EVEPGSGVR B*3501  30-38EPCGFEATY A*30101 supermotif  96-104 ASNGETLEK *predicted using rulesfound at the SYFPEITHI website(wysiwyg://35/http://134.2.96.221/scripts/hlaserver.dll/EpPredict.htm)and are based on the book “MHC Ligands and Peptide Motifs” by Rammensee,H. G., Bachmann, J. and S. Stevanovic. Chapman & Hall, New York, 1997.

TABLE 6 Predicted C35 HLA class II epitopes* Inclusive RestrictionSequence amino acids elements SGVRIVVEYCEPCGF 21-35 DRB1*0101 DRB1*0102DRB1*0301 DRB1*0401 DRB1*0404 DRB1*0405 DRB1*0410 DRB1*0421 DRB1*0701DRB1*0801 DRB1*0804 DRB1*0806 DRB1*1101 DRB1*1104 DRB1*1106 DRB1*1107DRB1*1305 DRB1*1307 DRB1*1321 DRB1*1501 DRB1*1502 DRB5*0101SRLGGTGAFEIEINGQLVF 57-75 DRB1*0101 DRB1*0102 DRB1*0301 DRB1*0401DRB1*0402 DRB1*0421 DRB1*0701 DRB1*0804 DRB1*0806 DRB1*1101 DRB1*1104DRB1*1106 DRB1*1305 DRB1*1321 DRB1*1501 DRB1*1502 DRB5*0101GAFEIEINGQLVFSKLENGGF 63-83 DRB1*0101 DRB1*0102 DRB1*0301 DRB1*0401DRB1*0402 DRB1*0404 DRB1*0405 DRB1*0410 DRB1*0421 DRB1*0701 DRB1*0804DRB1*0806 DRB1*1101 DRB1*1104 DRB1*1106 DRB1*1107 DRB1*1305 DRB1*1307DRB1*1311 DRB1*1321 DRB1*1501 DRB1*1502 DRB5*0101 FPYEKDLIEAIRRASNGETLE83-103 DRB1*0101 DRB1*0102 DRB1*0301 DRB1*0401 DRB1*0402 DRB1*0404DRB1*0405 DRB1*0410 DRB1*0421 DRB1*0701 DRB1*0801 DRB1*0802 DRB1*0804DRB1*0806 DRB1*1101 DRB1*1104 DRB1*1106 DRB1*1107 DRB1*1305 DRB1*1307DRB1*1311 DRB1*1321 DRB1*1501 DRB1*1502 DRB5*0101 *Class II MHC epitopespredicted using TEPITOPE software. Sturniolo, T., et al., “Generation oftissue-specific and promiscuous HLA ligand databases using DNAmicroarrays and virtual HLA class II matrices,”Nature Biotechnology 17:555-571 (1999).

In the present invention, “epitopes” refer to C35 polypeptide fragmentshaving antigenic or immunogenic activity in an animal, especially in ahuman, or that are capable of eliciting a T lymphocyte response in ananimal, preferably a human. A preferred embodiment of the presentinvention relates to a C35 polypeptide fragment comprising an epitope,as well as the polynucleotide encoding this fragment. A furtherpreferred embodiment of the present invention relates to a C35polypeptide fragment consisting of an epitope, as well as thepolynucleotide encoding this fragment. In specific preferred embodimentsof the present invention, the epitope comprises a C35 fragment listed inany of Tables 1-6. In another preferred embodiment of the presentinvention, the epitope consists of a C35 fragment listed in any ofTables 1-6. A region of a protein molecule to which an antibody can bindis defined as an “antigenic epitope.” In contrast, an “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse. (See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA81:3998-4002 (1983)). Thus, a further preferred embodiment of thepresent invention is an immunogenic C35 peptide fragment that is capableof eliciting a T cell response when bound to the peptide binding cleftof an MHC molecule. In a specific preferred embodiment, the immunogenicC35 peptide fragment comprises an epitope listed in any of Tables 1-6.In another preferred embodiment, the immunogenic C35 peptide fragmentconsists of an epitope listed in any of Tables 1-6. Further embodimentsof the invention are directed to pharmaceutical formulations and vaccinecompositions comprising said immunogenic C35 peptide fragments or thepolynucleotides encoding them.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA82:5131-5135 (1985) further described in U.S. Pat. No. 4,631,211.)

The sequence of peptide epitopes known to bind to specific MHC moleculescan be modified at the known peptide anchor positions in predictableways that act to increase MHC binding affinity. Such “epitopeenhancement” has been employed to improve the immunogenicity of a numberof different MHC class I or MHC class II binding peptide epitopes(Berzofsky, J. A. et al., Immunol. Rev. 170:151-72 (1999); Ahlers, J. D.et al., Proc. Natl. Acad. Sci. U.S.A. 94:10856-61 (1997); Overwijk, etal., J. Exp. Med. 188:277-86 (1998); Parkhurst, M. R. et al., J.Immunol. 157:2539-48 (1996)). Accordingly, a further embodiment of theinvention is directed to such enhanced C35 epitopes, and to thepolynucleotides encoding such enhanced epitomes.

In the present invention, antigenic epitopes preferably contain asequence of at least seven, more preferably at least nine, and mostpreferably between about 15 to about 30 amino acids. Antigenic epitopesare useful to raise antibodies, including monoclonal antibodies, thatspecifically bind the epitope. (See, for instance, Wilson et al., Cell37:767-778 (1984); Sutcliffe, J. G. et al., Science 219:660-666 (1983).)

Similarly, immunogenic epitopes can be used to induce B cells and Tcells according to methods well known in the art. (See Sutcliffe et al.,supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA82:910-914; and Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354(1985).) A preferred immunogenic epitope includes the secreted protein.The immunogenic epitopes may be presented together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse) or, if it is long enough (at least about 25 amino acids), withouta carrier. However, immunogenic epitopes comprising as few as 9 aminoacids have been shown to be sufficient to raise antibodies capable ofbinding to, at the very least, linear epitopes in a denaturedpolypeptide (e.g., in Western blotting.)

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to protein. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody.(Wahl et al., J. Nucl. Med. 24:316-325 (1983).) Thus, for someapplications these fragments are preferred, as well as the products of aFab or other immunoglobulin expression library. Moreover, antibodies ofthe present invention include chimeric, single chain, and humanizedantibodies.

Diagnostic and Therapeutic Uses of Antibodies

The present invention further relates to C35 antibodies, C35 antibodyfragments and antibody conjugates and single-chain immunotoxins reactivewith human carcinoma cells, particularly human breast and bladdercarcinoma cells.

Table 7 provides a list of C35-specific monoclonal antibodies that havebeen isolated and characterized for use in different applications.

TABLE 7 C35-Specific Murine Monoclonal Antibodies Fu- Hybrid- WesternFlow Immunohisto- sion oma ELISA Isotype Blot Cytometry chemistry alpha1F5 positive IgM Positive positive 1F7 positive IgM Positive 1F11positive IgM Positive 2D9 positive IgM positive Positive positive beta2G3 positive IgG1 2G8 positive 2G10 positive IgG3 2G11 positive IgG3 3F9positive IgG1 4D11 positive IgG1 4G3 positive IgG3 7C2 positive IgM 8B11positive IgM 8G2 positive IgM 10F4 positive IgG1 11B10 positive IgMpositive 12B10 positive 16C10 positive IgM 16F10 positive ELISA assay onbacterially-synthesized C35 blank = not determined

As used in this example, the following words or phrases have themeanings specified.

As used in this example, “joined” means to couple directly or indirectlyone molecule with another by whatever means, e.g., by covalent bonding,by non-covalent bonding, by ionic bonding, or by non-ionic bonding.Covalent bonding includes bonding by various linkers such as thioetherlinkers or thioester linkers. Direct coupling involves one moleculeattached to the molecule of interest. Indirect coupling involves onemolecule attached to another molecule not of interest which in turn isattached directly or indirectly to the molecule of interest.

As used in this example, “recombinant molecule” means a moleculeproduced by genetic engineering methods.

As used in this example, “fragment” is defined as at least a portion ofthe variable region of the immunoglobulin molecule which binds to itstarget, i.e. the antigen binding region. Some of the constant region ofthe immunoglobulin may be included.

As used in this example, an “immunoconjugate” means any molecule orligand such as an antibody or growth factor chemically or biologicallylinked to a cytotoxin, a radioactive agent, an anti-tumor drug or atherapeutic agent. The antibody or growth factor may be linked to thecytotoxin, radioactive agent, anti-tumor drug or therapeutic agent atany location along the molecule so long as it is able to bind itstarget. Examples of immunoconjugates include immunotoxins and antibodyconjugates.

As used in this example, “selectively killing” means killing those cellsto which the antibody binds.

As used in this example, examples of “carcinomas” include bladder,breast, colon, liver, lung, ovarian, and pancreatic carcinomas.

As used in this example, “immunotoxin” means an antibody or growthfactor chemically or biologically linked to a cytotoxin or cytotoxicagent.

As used in this example, an “effective amount” is an amount of theantibody, immunoconjugate, recombinant molecule which kills cells orinhibits the proliferation thereof.

As used in this example, “competitively inhibits” means being capable ofbinding to the same target as another molecule. With regard to anantibody, competitively inhibits mean that the antibody is capable ofrecognizing and binding the same antigen binding region to which anotherantibody is directed.

As used in this example, “antigen-binding region” means that part of theantibody, recombinant molecule, the fusion protein, or theimmunoconjugate of the invention which recognizes the target or portionsthereof.

As used in this example, “therapeutic agent” means any agent useful fortherapy including anti-tumor drugs, cytotoxins, cytotoxin agents, andradioactive agents.

As used in this example, “anti-tumor drug” means any agent useful tocombat cancer including, but not limited to, cytotoxins and agents suchas antimetabolites, alkylating agents, anthracyclines, antibiotics,antimitotic agents, procarbazine, hydroxyurea, asparaginase,corticosteroids, mytotane (O,P′-(DDD)), interferons and radioactiveagents.

As used in this example, “a cytotoxin or cytotoxic agent” means anyagent that is detrimental to cells. Examples include taxol, cytochalasinB, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicine, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

As used in this example, “radioisotope” includes any radioisotope whichis effective in destroying a tumor. Examples include, but are notlimited to, cobalt-60 and X-rays. Additionally, naturally occurringradioactive elements such as uranium, radium, and thorium whichtypically represent mixtures of radioisotopes, are suitable examples ofa radioactive agent.

As used in this example, “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular or subcutaneous administration, or theimplantation of a slow-release device such as a miniosmotic pump, to thesubject.

As used in this example, “directly” means the use of antibodies coupledto a label. The specimen is incubated with the labeled antibody, unboundantibody is removed by washing, and the specimen may be examined.

As used in this example, “indirectly” means incubating the specimen withan unconjugated antibody, washing and incubating with afluorochrome-conjugated antibody. The second or “sandwich” antibody thusreveals the presence of the first.

As used in this example “reacting” means to recognize and bind thetarget. The binding may be non-specific. Specific binding is preferred.

As used in this example, “curing” means to provide substantiallycomplete tumor regression so that the tumor is not palpable for a periodof time, i.e., >/=10 tumor volume doubling delays (TVDD=the time in daysthat it takes for control tumors to double in size).

As used in this example, “tumor targeted antibody” means any antibodywhich recognizes the C35 antigen on tumor (i.e., cancer) cells.

As used in this example, “inhibit proliferation” means to interfere withcell growth by whatever means.

As used in this example, “mammalian tumor cells” include cells fromanimals such as human, ovine, porcine, murine, bovine animals.

As used in this example, “pharmaceutically acceptable carrier” includesany material which when combined with the antibody retains theantibody's immunogenicity and is non-reactive with the subject's immunesystems. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsion, and various types ofwetting agents. Other carriers may also include sterile solutions,tablets including coated tablets and capsules.

Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid or salts thereof, magnesiumor calcium stearate, talc, vegetable fats or oils, gums, glycols, orother known excipients. Such carriers may also include flavor and coloradditives or other ingredients. Compositions comprising such carriersare formulated by well known conventional methods.

The present invention relates to C35 antibodies that are highly specificfor carcinoma cells. More particularly, the antibodies react with arange of carcinomas such as breast, bladder, lung, ovary and coloncarcinomas, while showing none or limited reactivity with normal humantissues or other types of tumors such as, for example, sarcomas orlymphomas.

The term “C35 antibody” as used herein includes whole, intact polyclonaland monoclonal antibody materials, and chimeric antibody molecules. TheC35 antibody described above includes any fragments thereof containingthe active antigen-binding region of the antibody such as Fab, F(ab′)2and Fv fragments, using techniques well established in the art [see,e.g., Rousseaux et al., “Optimal Conditions For The Preparation ofProteolytic Fragments From Monoclonal IgG of Different Rat IgGSubclasses”, in Methods Enzymol., 121:663-69 (Academic Press 1986)]. TheC35 antibody of the invention also includes fusion proteins.

Also included within the scope of the invention are anti-idiotypicantibodies to the C35 antibody of the invention. These anti-idiotypicantibodies can be produced using the C35 antibody and/or the fragmentsthereof as immunogen and are useful for diagnostic purposes in detectinghumoral response to tumors and in therapeutic applications, e.g., in avaccine, to induce an anti-tumor response in patients [see, e.g., Nepomet al., “Anti-Idiotypic Antibodies And The Induction Of Specific TumorImmunity”, in Cancer And Metastasis Reviews, 6:487-501 (1987)].

In addition, the present invention encompasses antibodies that arecapable of binding to the same antigenic determinant as the C35antibodies and competing with the antibodies for binding at that site.These include antibodies having the same antigenic specificity as theC35 antibodies but differing in species origin, isotype, bindingaffinity or biological functions (e.g., cytotoxicity). For example,class, isotype and other variants of the antibodies of the inventionhaving the antigen-binding region of the C35 antibody can be constructedusing recombinant class-switching and fusion techniques known in the art[see, e.g., Thammana et al., “Immunoglobulin Heavy Chain Class SwitchFrom IgM to IgG In A Hybridoma”, Eur. J. Immunol., 13:614 (1983); Spiraet al., “The Identification Of Monoclonal Class Switch Variants BySubselection And ELISA Assay”, J. Immunol. Meth. 74:307-15 (1984);Neuberger et al., “Recombinant Antibodies Possessing Novel EffectorFunctions”, Nature 312: 614-608 (1984); and Oi et al., “ChimericAntibodies”, Biotechniques 4 (3):214-21 (1986)]. Thus, other chimericantibodies or other recombinant antibodies (e.g., fusion proteinswherein the antibody is combined with a second protein such as alymphokine or a tumor inhibitory growth factor) having the same bindingspecificity as the C35-specific antibodies fall within the scope of thisinvention.

Genetic engineering techniques known in the art may be used as describedherein to prepare recombinant immunotoxins produced by fusing antigenbinding regions of antibody C35 to a therapeutic or cytotoxic agent atthe DNA level and producing the cytotoxic molecule as a chimericprotein. Examples of therapeutic agents include, but are not limited to,antimetabolites, alkylating agents, anthracyclines, antibiotics, andanti-mitotic agents. Antimetabolites include methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine.Alkylating agents include mechlorethamine, thioepa chlorambucil,melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide,busulfan, dibromomannitol, streptozotocin, mitomycin C, andcis-dichlorodiamine platinum (II) (DDP) cisplatin. Anthracyclinesinclude daunorubicin (formerly daunomycin) and doxorubicin (alsoreferred to herein as adriamycin). Additional examples includemitozantrone and bisantrene. Antibiotics include dactinomycin (formerlyactinomycin), bleomycin, mithramycin, and anthramycin (AMC). Antimytoticagents include vincristine and vinblastine (which are commonly referredto as vinca alkaloids). Other cytotoxic agents include procarbazine,hydroxyurea, asparaginase, corticosteroids, mytotane (O,P′-(DDD)),interferons. Further examples of cytotoxic agents include, but are notlimited to, ricin, doxorubicin, taxol, cytochalasin B, gramicidin D,ethidium bromide, etoposide, tenoposide, colchicine, dihydroxy anthracindione, 1-dehydrotestosterone, and glucocorticoid.

Clearly analogs and homologs of such therapeutic and cytotoxic agentsare encompassed by the present invention. For example, thechemotherapeutic agent aminopterin has a correlative improved analognamely methotrexate. Further, the improved analog of doxorubicin is anFe-chelate. Also, the improved analog for 1-methylnitrosourea islomustine. Further, the improved analog of vinblastine is vincristine.Also, the improved analog of mechlorethamine is cyclophosphamide.

Recombinant immunotoxins, particularly single-chain immunotoxins, havean advantage over drug/antibody conjugates in that they are more readilyproduced than these conjugates, and generate a population of homogenousmolecules, i.e. single peptides composed of the same amino acidresidues. The techniques for cloning and expressing DNA sequencesencoding the amino acid sequences corresponding to C35 single-chainimmunotoxins, e.g synthesis of oligonucleotides, PCR, transformingcells, constructing vectors, expression systems, and the like arewell-established in the art, and most practitioners are familiar withthe standard resource materials for specific conditions and procedures[see, e.g. Sambrook et al., eds., Molecular Cloning, A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)].

The following include preferred embodiments of the immunoconjugates ofthe invention. Other embodiments which are known in the art areencompassed by the invention. The invention is not limited to thesespecific immunoconjugates, but also includes other immunoconjugatesincorporating antibodies and/or antibody fragments according to thepresent invention.

The conjugates comprise at least one drug molecule connected by a linkerof the invention to a targeting ligand molecule that is reactive withthe desired target cell population. The ligand molecule can be animmunoreactive protein such as an antibody, or fragment thereof, anon-immunoreactive protein or peptide ligand such as bombesin or, abinding ligand recognizing a cell associated receptor such as a lectinor steroid molecule.

Further, because the conjugates of the invention can be used formodifying a given biological response, the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon,beta-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

The preferred drugs for use in the present invention are cytotoxicdrugs, particularly those which are used for cancer therapy. Such drugsinclude, in general, alkylating agents, anti-proliferative agents,tubulin binding agents and the like. Preferred classes of cytotoxicagents include, for example, the anthracycline family of drugs, thevinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides,the pteridine family of drugs, diynenes, and the podophyllotoxins.Particularly useful members of those classes include, for example,adriamycin, caminomycin, daunorubicin, aminopterin, methotrexate,methopterin, dichloromethotrexate, mitomycin C, porfiromycin,5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin,or podophyllotoxin derivatives such as etoposide or etoposide phosphate,melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosineand the like. As noted previously, one skilled in the art may makechemical modifications to the desired compound in order to makereactions of that compound more convenient for purposes of preparingconjugates of the invention.

As noted, one skilled in the art will appreciate that the invention alsoencompasses the use of antigen recognizing immunoglobulin fragments.Such immunoglobulin fragments may include, for example, the Fab′,F(ab′)2, F[v] or Fab fragments, or other antigen recognizingimmunoglobulin fragments. Such immunoglobulin fragments can be prepared,for example, by proteolytic enzyme digestion, for example, by pepsin orpapain digestion, reductive alkylation, or recombinant techniques. Thematerials and methods for preparing such immunoglobulin fragments arewell-known to those skilled in the art. See generally, Parham, J.Immunology, 131:2895 (1983); Lamoyi et al., J. Immunological Methods56:235 (1983); Parham, id., 53:133 (1982); and Matthew et al., id.,50:239 (1982).

The immunoglobulin can be a “chimeric antibody” as that term isrecognized in the art. Also, the immunoglobulin may be a “bifunctional”or “hybrid” antibody, that is, an antibody which may have one arm havinga specificity for one antigenic site, such as a tumor associated antigenwhile the other arm recognizes a different target, for example, a haptenwhich is, or to which is bound, an agent lethal to the antigen-bearingtumor cell. Alternatively, the bifunctional antibody may be one in whicheach arm has specificity for a different epitope of a tumor associatedantigen of the cell to be therapeutically or biologically modified. Inany case, the hybrid antibodies have a dual specificity, preferably withone or more binding sites specific for the hapten of choice or one ormore binding sites specific for a target antigen, for example, anantigen associated with a tumor, an infectious organism, or otherdisease state.

Biological bifunctional antibodies are described, for example, inEuropean Patent Publication, EPA 0 105 360, to which those skilled inthe art are referred. Such hybrid or bifunctional antibodies may bederived, as noted, either biologically, by cell fusion techniques, orchemically, especially with cross-linking agents or disulfidebridge-forming reagents, and may be comprised of whose antibodies and/orfragments thereof. Methods for obtaining such hybrid antibodies aredisclosed, for example, in PCT application WO83/03679, published Oct.27, 1983, and published European Application EPA 0 217 577, publishedApr. 8, 1987. Particularly preferred bifunctional antibodies are thosebiologically prepared from a “polydome” or “quadroma” or which aresynthetically prepared with cross-linking agents such asbis-(maleimideo)-methyl ether (“BMME”), or with other cross-linkingagents familiar to those skilled in the art.

In addition the immunoglobulin may be a single chain antibody (“SCA”).These may consist of single chain Fv fragments (“scFv”) in which thevariable light (“V[L]”) and variable heavy (“V[H]”) domains are linkedby a peptide bridge or by disulfide bonds. Also, the immunoglobulin mayconsist of single V[H] domains (dAbs) which possess antigen-bindingactivity. See, e.g., G. Winter and C. Milstein, Nature 349:295 (1991);R. Glockshuber et al., Biochemistry 29: 1362 (1990); and, E. S. Ward etal., Nature 341: 544 (1989).

Especially preferred for use in the present invention are chimericmonoclonal antibodies, preferably those chimeric antibodies havingspecificity toward a tumor associated antigen. As used in this example,the term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e. binding region, from one source or species andat least a portion of a constant region derived from a different sourceor species, usually prepared by recombinant DNA techniques. Chimericantibodies comprising a murine variable region and a human constantregion are preferred in certain applications of the invention,particularly human therapy, because such antibodies are readily preparedand may be less immunogenic than purely murine monoclonal antibodies.Such murine/human chimeric antibodies are the product of expressedimmunoglobulin genes comprising DNA segments encoding murineimmunoglobulin variable regions and DNA segments encoding humanimmunoglobulin constant regions. Other forms of chimeric antibodiesencompassed by the invention are those in which the class or subclasshas been modified or changed from that of the original antibody. Such“chimeric” antibodies are also referred to as “class-switchedantibodies”. Methods for producing chimeric antibodies involveconventional recombinant DNA and gene transfection techniques now wellknown in the art. See, e.g., Morrison, S. L. et al., Proc. Nat'l Acad.Sci., 81, 6851 (1984).

Encompassed by the term “chimeric antibody” is the concept of “humanizedantibody”, that is those antibodies in which the framework or“complementarity” determining regions (“CDR”) have been modified tocomprise the CDR of an immunoglobulin of different specificity ascompared to that of the parent immunoglobulin. In a preferredembodiment, a murine CDR is grafted into the framework region of a humanantibody to prepare the “humanized antibody”. See, e.g., L. Riechmann etal., Nature 332: 323 (1988); M. S. Neuberger et al., Nature 314: 268(1985). Particularly preferred CDR'S correspond to those representingsequences recognizing the antigens noted above for the chimeric andbifunctional antibodies. The reader is referred to the teaching of EPA 0239 400 (published Sep. 30, 1987), for its teaching of CDR modifiedantibodies.

One skilled in the art will recognize that a bifunctional-chimericantibody can be prepared which would have the benefits of lowerimmunogenicity of the chimeric or humanized antibody, as well as theflexibility, especially for therapeutic treatment, of the bifunctionalantibodies described above. Such bifunctional-chimeric antibodies can besynthesized, for instance, by chemical synthesis using cross-linkingagents and/or recombinant methods of the type described above. In anyevent, the present invention should not be construed as limited in scopeby any particular method of production of an antibody whetherbifunctional, chimeric, bifunctional-chimeric, humanized, or anantigen-recognizing fragment or derivative thereof.

In addition, the invention encompasses within its scope immunoglobulins(as defined above) or immunoglobulin fragments to which are fused activeproteins, for example, an enzyme of the type disclosed in Neuberger, etal., PCT application, WO86/01533, published Mar. 13, 1986. Thedisclosure of such products is incorporated herein by reference.

As noted, “bifunctional”, “fused”, “chimeric” (including humanized), and“bifunctional-chimeric” (including humanized) antibody constructionsalso include, within their individual contexts constructions comprisingantigen recognizing fragments. As one skilled in the art will recognize,such fragments could be prepared by traditional enzymatic cleavage ofintact bifunctional, chimeric, humanized, or chimeric-bifunctionalantibodies. If, however, intact antibodies are not susceptible to suchcleavage, because of the nature of the construction involved, the notedconstructions can be prepared with immunoglobulin fragments used as thestarting materials; or, if recombinant techniques are used, the DNAsequences, themselves, can be tailored to encode the desired “fragment”which, when expressed, can be combined in vivo or in vitro, by chemicalor biological means, to prepare the final desired intact immunoglobulin“fragment”. It is in this context, therefore, that the term “fragment”is used.

Furthermore, as noted above, the immunoglobulin (antibody), or fragmentthereof, used in the present invention may be polyclonal or monoclonalin nature. Monoclonal antibodies are the preferred immunoglobulins,however. The preparation of such polyclonal or monoclonal antibodies nowis well known to those skilled in the art who, of course, are fullycapable of producing useful immunoglobulins which can be used in theinvention. See, e.g., G. Kohler and C. Milstein, Nature 256: 495 (1975).In addition, hybridomes and/or monoclonal antibodies which are producedby such hybridomas and which are useful in the practice of the presentinvention are publicly available from sources such as the American TypeCulture Collection (“ATCC”) 10801 University Blvd., Manassas, Va. 20110.

Particularly preferred monoclonal antibodies for use in the presentinvention are those which recognize tumor associated antigens.

Diagnostic Techniques

Serologic diagnostic techniques involve the detection and quantitiationof tumor-associated antigens that have been secreted or “shed” into theserum or other biological fluids of patients thought to be sufferingfrom carcinoma. Such antigens can be detected in the body fluids usingtechniques known in the art such as radioimmunoassays (RIA) orenzyme-linked immunosorbent assays (ELISA) wherein an antibody reactivewith the “shed” antigen is used to detect the presence of the antigen ina fluid sample [see, e.g., Uotila et al., “Two-Site Sandwich ELISA WithMonoclonal Antibodies To Human AFP”, J. Immunol. Methods, 42:11 (1981)and Allum et al., supra at pp. 48-51]. These assays, using the C35antibodies disclosed herein, can therefore be used for the detection inbiological fluids of the antigen with which the C35 antibodies react andthus the detection of human carcinoma in patients. Thus, it is apparentfrom the foregoing that the C35 antibodies of the invention can be usedin most assays involving antigen-antibody reactions. These assaysinclude, but are not limited to, standard RIA techniques, both liquidand solid phase, as well as ELISA assays, ELISPOT, immunofluorescencetechniques, and other immunocytochemical assays [see, e.g., Sikora etal. (eds.), Monoclonal Antibodies, pp. 32-52 (Blackwell ScientificPublications 1984)].

The invention also encompasses diagnostic kits for carrying out theassays described above. In one embodiment, the diagnostic kit comprisesthe C35 monoclonal antibody, fragments thereof, fusion proteins orchimeric antibody of the invention, and a conjugate comprising aspecific binding partner for the C35 antibody and a label capable ofproducing a detectable signal. The reagents can also include ancillaryagents such as buffering agents and protein stabilizing agents (e.g.,polysaccharides). The diagnostic kit can further comprise, wherenecessary, other components of the signal-producing system includingagents for reducing background interference, control reagents or anapparatus or container for conducting the test.

In another embodiment, the diagnostic kit comprises a conjugate of theC35 antibodies of the invention and a label capable of producing adetectable single. Ancillary agents as mentioned above can also bepresent.

The C35 antibody of the invention is also useful for in vivo diagnosticapplications for the detection of human carcinomas. One such approachinvolves the detection of tumors in vivo by tumor imaging techniques.According to this approach, the C35 antibody is labeled with anappropriate imaging reagent that produces a detectable signal. Examplesof imaging reagents that can be used include, but at not limited to,radiolabels such as <131> I, <111> In, <123> I, <99 m> Tc, <32> P, <125>I, <3> H, and <14> C, fluorescent labels such as fluorescein andrhodamine, and chemiluninescers such as luciferin. The antibody can belabeled with such reagents using techniques known in the art. Forexample, see Wensel and Meares, Radioimmunoimaging AndRadioimmunotherapy, Elsevier, N.Y. (1983) for techniques relating to theradiolabeling of antibodies [see also, Colcher et al., “Use OfMonoclonal Antibodies As Radiopharmaceuticals For The Localization OfHuman Carcinoma Xenografts In Athymic Mice”, Meth. Enzymol. 121:802-16(1986)].

In the case of radiolabeled antibody, the antibody is administered tothe patient, localizes to the tumor bearing the antigen with which theantibody reacts, and is detected or “imaged” in vivo using knowntechniques such as radionuclear scanning using, e.g., a gamma camera oremission tomography [see, e.g., Bradwell et al., “Developments InAntibody Imaging”, in Monoclonal Antibodies For Cancer Detection AndTherapy, Baldwin et al. (eds.), pp. 65-85 (Academic Press 1985)]. Theantibody is administered to the patient in a pharmaceutically acceptablecarrier such as water, saline, Ringer's solution, Hank's solution ornonaqueous carriers such as fixed oils. The carrier may also containsubstances that enhance isotonicity and chemical stability of theantibody such as buffers or preservatives. The antibody formulation isadministered, for example, intravenously, at a dosage sufficient toprovide enough gamma emission to allow visualization of the tumor targetsite. Sufficient time should be allowed between administration of theantibody and detection to allow for localization to the tumor target.For a general discussion of tumor imaging, see Allum et al., supra atpp. 51-55.

Therapeutic Applications of C35 Antibodies

The properties of the C35 antibody suggest a number of in vivotherapeutic applications.

First, the C35 antibody can be used alone to target and kill tumor cellsin vivo. The antibody can also be used in conjunction with anappropriate therapeutic agent to treat human carcinoma. For example, theantibody can be used in combination with standard or conventionaltreatment methods such as chemotherapy, radiation therapy or can beconjugated or linked to a therapeutic drug, or toxin, as well as to alymphokine or a tumor-inhibitory growth factor, for delivery of thetherapeutic agent to the site of the carcinoma.

Techniques for conjugating such therapeutic agents to antibodies arewell known [see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982)].

Alternatively, the C35 antibody can be coupled to high-energy radiation,e.g., a radioisotope such as <131> I, which, when localized at the tumorsite, results in a killing of several cell diameters [see, e.g., Order,“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16(Academic Press 1985)]. According to yet another embodiment, the C35antibody can be conjugated to a second antibody to form an antibodyheteroconjugate for the treatment of tumor cells as described by Segalin U.S. Pat. No. 4,676,980.

Still other therapeutic applications for the C35 antibody of theinvention include conjugation or linkage, e.g., by recombinant DNAtechniques, to an enzyme capable of converting a prodrug into acytotoxic drug and the use of that antibody-enzyme conjugate incombination with the prodrug to convert the prodrug to a cytotoxic agentat the tumor site [see, e.g., Senter et al., “Anti-Tumor Effects OfAntibody-alkaline Phosphatase”, Proc. Natl. Acad. Sci. USA, 85:4842-46(1988); “Enhancement of the in vitro and in vivo Antitumor Activites ofPhosphorylated Mitocycin C and Etoposide Derivatives by MonoclonalAntibody-Alkaline Phosphatase Conjugates”, Cancer Research 49:5789-5792(1989); and Senter, “Activation of Prodrugs by Antibody-EnzymeConjugates: A New Approach to Cancer Therapy,” FASEB J. 4:188-193(1990)].

Still another therapeutic use for the C35 antibody involves use, eitherin the presence of complement or as part of an antibody-drug orantibody-toxin conjugate, to remove tumor cells from the bone marrow ofcancer patients. According to this approach, autologous bone marrow maybe purged ex vivo by treatment with the antibody and the marrow infusedback into the patient [see, e.g., Ramsay et al., “Bone Marrow PurgingUsing Monoclonal Antibodies”, J. Clin. Immunol., 8(2):81-88 (1988)].

Furthermore, chimeric C35, recombinant immunotoxins and otherrecombinant constructs of the invention containing the specificity ofthe antigen-binding region of the C35 monoclonal antibody, as describedearlier, may be used therapeutically. For example, the single-chainimmunotoxins of the invention, may be used to treat human carcinoma invivo.

Similarly, a fusion protein comprising at least the antigen-bindingregion of the C35 antibody joined to at least a functionally activeportion of a second protein having anti-tumor activity, e.g., alymphokine or oncostatin can be used to treat human carcinoma in vivo.Furthermore, recombinant techniques known in the art can be used toconstruct bispecific antibodies wherein one of the binding specificitiesof the antibody is that of C35, while the other binding specificity ofthe antibody is that of a molecule other than C35.

Finally, anti-idiotypic antibodies of the C35 antibody may be usedtherapeutically in active tumor immunization and tumor therapy [see,e.g., Hellstrom et al., “Immunological Approaches To Tumor Therapy:Monoclonal Antibodies, Tumor Vaccines, And Anti-Idiotypes”, inCovalently Modified Antigens And Antibodies In Diagnosis And Therapy,supra at pp. 35-41].

The present invention provides a method for selectively killing tumorcells expressing the antigen that specifically binds to the C35monoclonal antibody or functional equivalent. This method comprisesreacting the immunoconjugate (e.g. the immunotoxin) of the inventionwith said tumor cells. These tumor cells may be from a human carcinoma.

Additionally, this invention provides a method of treating carcinomas(for example human carcinomas) in vivo. This method comprisesadministering to a subject a pharmaceutically effective amount of acomposition containing at least one of the immunoconjugates (e.g. theimmunotoxin) of the invention.

In accordance with the practice of this invention, the subject may behuman, equine, porcine, bovine, murine, canine, feline, and aviansubjects. Other warm blooded animals are also included in thisinvention.

The present invention also provides a method for curing a subjectsuffering from a cancer. The subject may be a human, dog, cat, mouse,rat, rabbit, horse, goat, sheep, cow, chicken. The cancer may beidentified as a breast, bladder, retinoblastoma, papillarycystadenocarcinoma of the ovary, Wilm's tumor, or small cell lungcarcinoma and is generally characterized as a group of cells havingtumor associated antigens on the cell surface. This method comprisesadministering to the subject a cancer killing amount of a tumor targetedantibody joined to a cytotoxic agent. Generally, the joining of thetumor targeted antibody with the cytotoxic agent is made underconditions which permit the antibody so joined to bind its target on thecell surface. By binding its target, the tumor targeted antibody actsdirectly or indirectly to cause or contribute to the killing of thecells so bound thereby curing the subject.

Also provided is a method of inhibiting the proliferation of mammaliantumor cells which comprises contacting the mammalian tumor cells with asufficient concentration of the immunoconjugate of the invention so asto inhibit proliferation of the mammalian tumor cells.

The subject invention further provides methods for inhibiting the growthof human tumor cells, treating a tumor in a subject, and treating aproliferative type disease in a subject. These methods compriseadministering to the subject an effective amount of the composition ofthe invention.

It is apparent therefore that the present invention encompassespharmaceutical compositions, combinations and methods for treating humancarcinomas. For example, the invention includes pharmaceuticalcompositions for use in the treatment of human carcinomas comprising apharmaceutically effective amount of a C35 antibody and apharmaceutically acceptable carrier.

The compositions may contain the C35 antibody or antibody fragments,either unmodified, conjugated to a therapeutic agent (e.g., drug, toxin,enzyme or second antibody) or in a recombinant form (e.g., chimeric C35,fragments of chimeric C35, bispecific C35 or single-chain immunotoxinC35). The compositions may additionally include other antibodies orconjugates for treating carcinomas (e.g., an antibody cocktail).

The antibody, antibody conjugate and immunotoxin compositions of theinvention can be administered using conventional modes of administrationincluding, but not limited to, intravenous, intraperitoneal, oral,intralymphatic or administration directly into the tumor. Intravenousadministration is preferred.

The compositions of the invention may be in a variety of dosage formswhich include, but are not limited to, liquid solutions or suspension,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application.

The compositions of the invention also preferably include conventionalpharmaceutically acceptable carriers and adjuvants known in the art suchas human serum albumin, ion exchangers, alumina, lecithin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,and salts or electrolytes such as protamine sulfate.

The most effective mode of administration and dosage regimen for thecompositions of this invention depends upon the severity and course ofthe disease, the patient's health and response to treatment and thejudgment of the treating physician. Accordingly, the dosages of thecompositions should be titrated to the individual patient. Nevertheless,an effective dose of the compositions of this invention may be in therange of from about 1 to about 2000 mg/kg.

The molecules described herein may be in a variety of dosage forms whichinclude, but are not limited to, liquid solutions or suspensions,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application.

The most effective mode of administration and dosage regimen for themolecules of the present invention depends upon the location of thetumor being treated, the severity and course of the cancer, thesubject's health and response to treatment and the judgment of thetreating physician. Accordingly, the dosages of the molecules should betitrated to the individual subject.

The interrelationship of dosages for animals of various sizes andspecies and humans based on mg/kg of surface area is described byFreireich, E. J., et al. Cancer Chemother., Rep. 50 (4): 219-244 (1966).Adjustments in the dosage regimen may be made to optimize the tumor cellgrowth inhibiting and killing response, e.g., doses may be divided andadministered on a daily basis or the dose reduced proportionallydepending upon the situation (e.g., several divided doses may beadministered daily or proportionally reduced depending on the specifictherapeutic situation.

It would be clear that the dose of the composition of the inventionrequired to achieve cures may be further reduced with scheduleoptimization.

In accordance with the practice of the invention, the pharmaceuticalcarrier may be a lipid carrier. The lipid carrier may be a phospholipid.Further, the lipid carrier may be a fatty acid. Also, the lipid carriermay be a detergent. As used herein, a detergent is any substance thatalters the surface tension of a liquid, generally lowering it.

In one example of the invention, the detergent may be a nonionicdetergent. Examples of nonionic detergents include, but are not limitedto, polysorbate 80 (also known as Tween 80 or (polyoxyethylenesorbitanmonooleate), Brij, and Triton (for example Triton WR-1339 and TritonA-20).

Alternatively, the detergent may be an ionic detergent. An example of anionic detergent includes, but is not limited to, alkyltrimethylammoniumbromide.

Additionally, in accordance with the invention, the lipid carrier may bea liposome. As used in this application, a “liposome” is any membranebound vesicle which contains any molecules of the invention orcombinations thereof.

Vaccine Formulations

The C35 epitopes can be produced in quantity by recombinant DNA methodsand formulated with an adjuvant that promotes a cell-mediated immuneresponse. The present invention encompasses the expression of the C35polypeptides, or C35 epitopes (including cytotoxic or helper T celleliciting epitopes), in either eucaryotic or procaryotic recombinantexpression vectors; and the formulation of the same as immunogenicand/or antigenic compositions. Such compositions are described in, forexample, U.S. patent application Ser. No. 08/935,377, the entirecontents of which are incorporated herein by reference. In accordancewith the present invention, the recombinantly expressed C35 epitope maybe expressed, purified and formulated as a subunit vaccine. Preferably,the DNA encoding the C35 epitope may also be constructed into viralvectors, preferably pox virus, adenovirus, herpesvirus, and alphavirusvectors, for use in vaccines. In this regard, either a live recombinantviral vaccine, an inactivated recombinant viral vaccine, or a killedrecombinant viral vaccine can be formulated.

(i) Expression of C35 in Procaryotic and Eucaryotic Expression Systems

The present invention encompasses expression systems, both eucaryoticand procaryotic expression vectors, which may be used to express the C35epitope. The C35 epitope may be expressed in both truncated orfull-length forms, in particular for the formation of subunit vaccines.

The present invention encompasses the expression of nucleotide sequencesencoding the C35 polypeptide and immunologically equivalent fragments.Such immunologically equivalent fragments may be identified by makinganalogs of the nucleotide sequence encoding the identified epitopes thatare truncated at the 5′ and/or 3′ ends of the sequence and/or have oneor more internal deletions, expressing the analog nucleotide sequences,and determining whether the resulting fragments immunologically arerecognized by the epitope-specific T lymphocytes and induce acell-mediated immune response, or epitope-specific B lymphocytes forinductions of a humoral immune response.

The invention encompasses the DNA expression vectors that contain any ofthe foregoing coding sequences operatively associated with a regulatoryelement that directs expression of the coding sequences and geneticallyengineered host cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

The C35 epitope gene products or peptide fragments thereof, may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing the C35 epitope gene polypeptidesand peptides of the invention by expressing nucleic acid containingepitope gene sequences are described herein. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing epitope gene product coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques described in Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., (1989), Cold Spring Harbor Laboratory Press, andAusubel et al., 1989, supra. Alternatively, RNA capable of encodingglycoprotein epitope gene product sequences may be chemicallysynthesized using, for example, synthesizers. See, for example, thetechniques described in “Oligonucleotide Synthesis”, 1984, Gait, M. J.ed., IRL Press, Oxford, which is incorporated by reference herein in itsentirety.

The invention also encompasses nucleotide sequences that encode peptidefragments of the C35 epitope gene products. For example, polypeptides orpeptides corresponding to the extracellular domain of the C35 epitopemay be useful as “soluble” protein which would facilitate secretion,particularly useful in the production of subunit vaccines. The C35epitope gene product or peptide fragments thereof, can be linked to aheterologous epitope that is recognized by a commercially availableantibody is also included in the invention. A durable fusion protein mayalso be engineered; i.e., a fusion protein which has a cleavage sitelocated between the C35 epitope sequence and the heterologous proteinsequence, so that the selected C35 can be cleaved away from theheterologous moiety. For example, a collagenase cleavage recognitionconsensus sequence may be engineered between the C35 epitope protein orpeptide and the heterologous peptide or protein. The epitopic domain canbe released from this fusion protein by treatment with collagenase. In apreferred embodiment of the invention, a fusion protein ofglutathione-S-transferase and the C35 epitope protein may be engineered.

The C35 epitope proteins of the present invention for use in vaccinepreparations, in particular subunit vaccine preparations, aresubstantially pure or homogeneous. The protein is consideredsubstantially pure or homogeneous when at least 60 to 75% of the sampleexhibits a single polypeptide sequence. A substantially pure proteinwill preferably comprise 60 to 90% of a protein sample, more preferablyabout 95% and most preferably 99%. Methods which are well known to thoseskilled in the art can be used to determine protein purity orhomogeneity, such as polyacrylamide gel electrophoresis of a sample,followed by visualizing a single polypeptide band on a staining gel.Higher resolution may be determined using HPLC or other similar methodswell known in the art.

The present invention encompasses C35 polypeptides which are typicallypurified from host cells expressing recombinant nucleotide sequencesencoding these proteins. Such protein purification can be accomplishedby a variety of methods well known in the art. In a preferredembodiment, the C35 epitope protein of the present invention isexpressed as a fusion protein with glutathione-S-transferase. Theresulting recombinant fusion proteins purified by affinitychromatography and the epitope protein domain is cleaved away from theheterologous moiety resulting in a substantially pure protein sample.Other methods known to those skilled in the art may be used; see forexample, the techniques described in “Methods In Enzymology”, 1990,Academic Press, Inc., San Diego, “Protein Purification: Principles andPractice”, 1982, Springer-Verlag, New York, which are incorporated byreference herein in their entirety.

(ii) Eucaryotic and Procaryotic Expression Vectors

The present invention encompasses expression systems, both eucaryoticand procaryotic expression vectors, which may be used to express the C35epitope. A variety of host-expression vector systems may be utilized toexpress the C35 epitope gene of the invention. Such host-expressionsystems represent vehicles by which the C35 coding sequence may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the C35 nucleotide codingsequences, exhibit the C35 epitope gene product of the invention insitu. These include but are not limited to microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the C35 epitope gene product coding sequence; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing the C35 epitope gene product coding sequence; insectcell systems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the C35 epitope gene product coding sequence;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing C35 epitope gene product coding sequence; ormammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

(iii) Host Cells

The present invention encompasses the expression of the C35 epitope inanimal and insect cell lines. In a preferred embodiment of the presentinvention, the C35 epitope is expressed in a baculovirus vector in aninsect cell line to produce an unglycosylated antigen. In anotherpreferred embodiment of the invention, the C35 epitope is expressed in astably transfected mammalian host cell, e.g., CHO cell line to produce aglycosylated antigen. The C35 epitopes which are expressed recombinantlyby these cell lines may be formulated as subunit vaccines. The presentinvention is further directed to host cells that overexpress the C35gene product. The cell may be a host cell transiently or stabletransected or transformed with any suitable vector which includes apolynucleotide sequence encoding the C35 polypeptide or a fragmentthereof and suitable promoter and enhancer sequences to directoverexpression of the C35 gene product. However, the overexpressing cellmay also be a product of an insertion, for example via homologousrecombination, of a heterologous promoter or enhancer which will directoverexpression of the endogenous C35 gene. The term “overexpression”refers to a level of expression which is higher than a basal level ofexpression typically characterizing a given cell under otherwiseidentical conditions.

A host cell strain may be chosen which modulates the expression of theinserted sequences, or modifies and processes the C35 gene product inthe specific fashion desired.

Such modifications (e.g., glycosylation) and processing (e.g., cleavage)of protein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification of the foreign protein expressed. To this end,eucaryotic host cells which possess the cellular machinery for properprocessing of the primary transcript, glycosylation, phosphorylation,and prenylation of the C35 gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3 and WI38 cell lines.

For long term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe C35 target epitope may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines. This method mayadvantageously be used to engineer cell lines which express the C35epitope gene products. Such cell lines would be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the C35 epitope gene product.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30: 147).

Alternatively, any fusion protein may be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺-nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

(iv) Expression of C35 Epitope in Recombinant Viral Vaccines

In another embodiment of the present invention, either a liverecombinant viral vaccine or an inactivated recombinant viral vaccineexpressing the C35 epitope can be engineered. A live vaccine may bepreferred because multiplication in the host leads to a prolongedstimulus of similar kind and magnitude to that occurring in naturalinfections, and therefore, confers substantial, long-lasting immunity.Production of such live recombinant virus vaccine formulations may beaccomplished using conventional methods involving propagation of thevirus in cell culture or in the allantois of the chick embryo followedby purification.

In this regard, a variety of viruses may be genetically engineered toexpress the C35 epitope. For vaccine purposes, it may be required thatthe recombinant viruses display attenuation characteristics. Currentlive virus vaccine candidates for use in humans are either cold adapted,temperature sensitive, or attenuated. The introduction of appropriatemutations (e.g., deletions) into the templates used for transfection mayprovide the novel viruses with attenuation characteristics. For example,specific multiple missense mutations that are associated withtemperature sensitivity or cold adaptation can be made into deletionmutations and/or multiple mutations can be introduced into individualviral genes. These mutants should be more stable than the cold ortemperature sensitive mutants containing single point mutations andreversion frequencies should be extremely low. Alternatively,recombinant viruses with “suicide” characteristics may be constructed.Such viruses go through only one or a few rounds of replication in thehost.

For purposes of the invention, any virus may be used in accordance withthe present invention which: (a) displays an attenuated phenotype or maybe engineered to display attenuated characteristics; (b) displays atropism for mammals, in particular humans, or may be engineered todisplay such a tropism; and (c) may be engineered to express the C35epitope of the present invention.

Vaccinia viral vectors may be used in accordance with the presentinvention, as large fragments of DNA are easily cloned into its genomeand recombinant attenuated vaccinia variants have been described (Meyer,et al., 1991, J. Gen. Virol. 72:1031-1038). Orthomyxoviruses, includinginfluenza; Paramyxoviruses, including respiratory syncytial virus andSendai virus; and Rhabdoviruses may be engineered to express mutationswhich result in attenuated phenotypes (see U.S. Pat. No. 5,578,473,issued Nov. 26, 1996). These viral genomes may also be engineered toexpress foreign nucleotide sequences, such as the C35 epitopes of thepresent invention (see U.S. Pat. No. 5,166,057, issued Nov. 24, 1992,incorporated herein by reference in its entirety).

Reverse genetic techniques can be applied to manipulate negative andpositive strand RNA viral genomes to introduce mutations which result inattenuated phenotypes, as demonstrated in influenza virus, HerpesSimplex virus, cytomegalovirus and Epstein-Barr virus, Sindbis virus andpoliovirus (see Palese et al., 1996, Proc. Natl. Acad. Sci. USA93:11354-11358). These techniques may also be utilized to introduceforeign DNA, i.e., the C35 epitopes, to create recombinant viral vectorsto be used as vaccines in accordance with the present invention. See,for instance, U.S. patent application Ser. No. 08/935,377, the entirecontents of which are incorporated herein by reference. In addition,attenuated adenoviruses and retroviruses may be engineered to expressthe C35 epitope. Therefore, a wide variety of viruses may be engineeredto design the vaccines of the present invention, however, by way ofexample, and not by limitation, recombinant attenuated vaccinia vectorsexpressing the C35 epitope for use as vaccines are described herein.

In one embodiment, a recombinant modified vaccinia variant, ModifiedVirus Ankara (MVA) is used in a vaccine formulation. This modified virushas been passaged for 500 cycles in avian cells and is unable to undergoa full infectious cycle in mammalian cells (Meyer, et al., 1991, J. Gen.Virol. 72:1031-1038). When used as a vaccine, the recombinant virus goesthrough a single replication cycle and induces a sufficient level ofimmune response but does not go further in the human host and causedisease. Recombinant viruses lacking one or more of essential vacciniavirus genes are not able to undergo successive rounds of replication.Such defective viruses can be produced by co-transfecting vacciniavectors lacking a specific gene(s) required for viral replication intocell lines which permanently express this gene(s). Viruses lacking anessential gene(s) will be replicated in these cell lines but whenadministered to the human host will not be able to complete a round ofreplication. Such preparations may transcribe and translate—in thisabortive cycle—a sufficient number of genes to induce an immuneresponse.

Alternatively, larger quantities of the strains can be administered, sothat these preparations serve as inactivated (killed) virus, vaccines.For inactivated vaccines, it is preferred that the heterologous C35 geneproduct be expressed as a viral component, so that the C35 gene productis associated with the virion. The advantage of such preparations isthat they contain native proteins and do not undergo inactivation bytreatment with formalin or other agents used in the manufacturing ofkilled virus vaccines.

In another embodiment of the invention, inactivated vaccine formulationsare prepared using conventional techniques to “kill” the recombinantviruses. Inactivated vaccines are “dead” in the sense that theirinfectivity has been destroyed. Ideally, the infectivity of the virus isdestroyed without affecting immunogenicity. In order to prepareinactivated vaccines, the recombinant virus may be grown in cell cultureor in the allantois of the chick embryo, purified by zonalultracentrifugation, inactivated by formaldehyde or β-propiolactone, andpooled. The resulting vaccine is usually inoculated intramuscularly.

Inactivated viruses may be formulated with a suitable adjuvant in orderto enhance the immunological response. Such adjuvants may include butare not limited to mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols, polyanions;peptides; oligonucleotides, oil emulsions; and potentially useful humanadjuvants such as BCG and Corynebacterium parvum.

(v) Methods of Treatment and/or Vaccination

Since the C35 epitopes of the present invention can be produced in largeamounts, the antigen thus produced and purified has use in vaccinepreparations. The C35 epitope may be formulated into a subunit vaccinepreparation, or may be engineered into viral vectors and formulated intovaccine preparations. Alternatively, the DNA encoding the C35 epitopemay be administered directly as a vaccine formulation. The “naked”plasmid DNA once administered to a subject invades cells, is expressed,processed into peptide fragments, some of which can be presented inassociation with MHC molecules on the surface of the invaded cell, andelicits a cellular immune response so that T lymphocytes will attackcells displaying the C35 epitope. The C35 epitope also has utility indiagnostics, e.g., to detect or measure in a sample of body fluid from asubject the presence of tumors that express C35 or the presence ofantibodies or T cells that have been induced by C35 expressing tumor andthus to diagnose cancer and tumors and/or to monitor the cellular immuneresponse of the subject subsequent to vaccination.

The recombinant viruses of the invention can be used to treattumor-bearing mammals, including humans, to generate an immune responseagainst the tumor cells. The generation of an adequate and appropriateimmune response leads to tumor regression in vivo. Such “vaccines” canbe used either alone or in combination with other therapeutic regimens,including but not limited to chemotherapy, radiation therapy, surgery,bone marrow transplantation, etc. for the treatment of tumors. Forexample, surgical or radiation techniques could be used to debulk thetumor mass, after which, the vaccine formulations of the invention canbe administered to ensure the regression and prevent the progression ofremaining tumor masses or micrometastases in the body. Alternatively,administration of the “vaccine” can precede such surgical, radiation orchemotherapeutic treatment.

Alternatively, the recombinant viruses of the invention can be used toimmunize or “vaccinate” tumor-free subjects to prevent tumor formation.With the advent of genetic testing, it is now possible to predict asubject's predisposition for certain cancers. Such subjects, therefore,may be immunized using a recombinant vaccinia virus expressing the C35antigen.

The immunopotency of the C35 epitope vaccine formulations can bedetermined by monitoring the immune response in test animals followingimmunization or by use of any immunoassay known in the art. Generationof a cell-mediated and/or humoral immune response may be taken as anindication of an immune response. Test animals may include mice,hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., andeventually human subjects.

Suitable preparations of such vaccines include injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,suspension in, liquid prior to injection, may also be prepared. Thepreparation may also be emulsified, or the polypeptides encapsulated inliposomes. The active immunogenic ingredients are often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine preparation may also include minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and/or adjuvants which enhance the effectiveness ofthe vaccine.

Examples of adjuvants which may be effective, include, but are notlimited to: aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine,GM-CSF, QS-21 (investigational drug, Progenics Pharmaceuticals, Inc.),DETOX (investigational drug, Ribi Pharmaceuticals), BCG, and CpG richoligonucleotides.

The effectiveness of an adjuvant may be determined by measuring theinduction of the cellular immune response directed against the C35epitome.

The vaccines of the invention may be multivalent or univalent.Multivalent vaccines are made from recombinant viruses that direct theexpression of more than one antigen. Multivalent vaccines comprised ofmultiple T cell epitomes, both cytotoxic and helper, are preferred.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc.

Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent. Where thecomposition is administered by injection, an ampoule of sterile diluentcan be provided so that the ingredients may be mixed prior toadministration.

In a specific embodiment, a lyophilized C35 epitope of the invention isprovided in a first container; a second container comprises diluentconsisting of an aqueous solution of 50% glycerin, 0.25% phenol, and anantiseptic (e.g., 0.005% brilliant green).

Use of purified C35 antigens as vaccine preparations can be carried outby standard methods. For example, the purified C35 epitopes should beadjusted to an appropriate concentration, formulated with any suitablevaccine adjuvant and packaged for use. Suitable adjuvants may include,but are not limited to: mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols; polyanions;peptides; oil emulsions; alum, and MDP. The immunogen may also beincorporated into liposomes, or conjugated to polysaccharides and/orother polymers for use in a vaccine formulation. In instances where therecombinant antigen is a hapten, i.e., a molecule that is antigenic inthat it can react selectively with cognate antibodies, but notimmunogenic in that it cannot elicit an immune response, the hapten maybe covalently bound to a carrier or immunogenic molecule; for instance,a large protein such as serum albumin will confer immunogenicity to thehapten coupled to it. The hapten-carrier may be formulated for use as avaccine.

Many methods may be used to introduce the vaccine formulations describedabove into a patient. These include, but are not limited to, oral,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, transdermal, epidural, pulmonary, gastric, intestinal,rectal, vaginal, or urethral routes. When the method of treatment uses alive recombinant vaccinia vaccine formulation of the invention, it maybe preferable to introduce the formulation via the natural route ofinfection of the vaccinia virus, i.e., through a mucosal membrane orsurface, such as an oral, nasal, gastric, intestinal, rectal, vaginal orurethral route, or through the skin. To induce a CTL response, themucosal route of administration may be through an oral or nasalmembrane. Alternatively, an intramuscular or intraperitoneal route ofadministration may be used. Preferably, a dose of 10⁶-10⁷ PFU (plaqueforming units) of cold adapted recombinant vaccinia virus is given to ahuman patient.

The precise dose of vaccine preparation to be employed in theformulation will also depend on the route of administration, and thenature of the patient, and should be decided according to the judgmentof the practitioner and each patient's circumstances according tostandard clinical techniques. An effective immunizing amount is thatamount sufficient to produce an immune response to the antigen in thehost to which the vaccine preparation is administered.

Where subsequent or booster doses are required, a modified vacciniavirus such as MVA can be selected as the parental virus used to generatethe recombinant. Alternatively, another virus, e.g., adenovirus, canarypox virus, or a subunit preparation can be used to boost. Immunizationand/or cancer immunotherapy may be accomplished using a combinedimmunization regimen, e.g., immunization with a recombinant vacciniaviral vaccine of the invention and a boost of a recombinant adenoviralvaccine. In such an embodiment, a strong secondary CD8⁺ T cell responseis induced after priming and boosting with different viruses expressingthe same epitope (for such methods of immunization and boosting, see,e.g., Murata et al., Cellular Immunol. 173:96-107). For example, apatient is first primed with a vaccine formulation of the inventioncomprising a recombinant vaccinia virus expressing an epitope, e.g., aselected tumor-associated antigen or fragment thereof. The patient isthen boosted, e.g., 21 days later, with a vaccine formulation comprisinga recombinant virus other than vaccinia expressing the same epitope.Such priming followed by boosting induces a strong secondary T cellresponse. Such a priming and boosting immunization regimen is preferablyused to treat a patient with a tumor, metastasis or neoplastic growthexpressing the tumor associate, e.g., C35, antigen.

In yet another embodiment, the recombinant vaccinia viruses can be usedas a booster immunization subsequent to a primary immunization withinactivated tumor cells, a subunit vaccine containing the C35 antigen orits epitope, or another recombinant viral vaccine, e.g., adenovirus,canary pox virus, or MVA.

In an alternate embodiment, recombinant vaccinia virus encoding C35epitopes or fragment thereof may be used in adoptive immunotherapeuticmethods for the activation of T lymphocytes that are histocompatiblewith the patient and specific for the C35 antigen (for methods ofadoptive immunotherapy, see, e.g., Rosenberg, U.S. Pat. No. 4,690,915,issued Sep. 1, 1987; Zarling, et al., U.S. Pat. No. 5,081,029, issuedJan. 14, 1992). Such T lymphocytes may be isolated from the patient or ahistocompatible donor. The T lymphocytes are activated in vitro byexposure to the recombinant vaccinia virus of the invention. Activated Tlymphocytes are expanded and inoculated into the patient in order totransfer T cell immunity directed against the C35 antigen epitome.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers comprising one or more of the ingredients of thevaccine formulations of the invention. Associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

Cancer Diagnosis and Prognosis

There are two classes of genes affecting tumor development. Genesinfluencing the cancer phenotype that act directly as a result ofchanges (e.g., mutation) at the DNA level, such as BRCA1, BRCA2, andp53, are one class of genes. Another class of genes affect the phenotypeby modulation at the expression level. Development of breast cancer andsubsequent malignant progression is associated with alterations of avariety of genes of both classes. Identification of quantitative changesin gene expression that occur in the malignant mammary gland, ifsufficiently characterized, may yield novel molecular markers which maybe useful in the diagnosis and treatment of human breast cancer.

The present inventors have identified a new breast cancer marker, C35,that is differentially expressed in primary infiltrating intraductalmammary carcinoma cells. Low expression levels of C35 in normal mammaryepithelial cells suggest that overexpression of C35 indicates breastcancer malignant progression. It is possible that C35 may also beoverexpressed in tumors of certain other tissue types including bladderand lung.

The present inventors have demonstrated that certain tissues in mammalswith cancer express significantly enhanced levels of the C35 protein andmRNA encoding the C35 protein when compared to a corresponding“standard” mammal, i.e., a mammal of the same species not having thecancer. Further, it is believed that enhanced levels of the C35 protein,or of antibodies or lymphocytes specific for the C35 protein, can bedetected in certain body fluids (e.g., sera, plasma, urine, and spinalfluid) from mammals with cancer when compared to sera from mammals ofthe same species not having the cancer. Thus, the present inventionprovides a diagnostic method useful for tumor diagnosis, which involvesassaying the expression level of the gene encoding the C35 protein inmammalian cells or body fluid and comparing the gene expression levelwith a standard C35 gene expression level, whereby an increase in thegene expression level over the standard is indicative of certain tumors.Alternatively, the expression levels of antibodies or lymphocytesspecific for C35 protein or C35 polypeptides can be determined in bloodor other body fluids and compared with a standard of expression ofC35-specific antibodies or lymphocytes.

Where a tumor diagnosis has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting enhanced C35 gene expression may experiencea worse clinical outcome relative to patients expressing the gene at alower level.

By “assaying the expression level of the gene encoding the C35 protein”is intended qualitatively or quantitatively measuring or estimating thelevel of the C35 protein or the level of the mRNA encoding the C35protein in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level or mRNA level) orrelatively (e.g., by comparing to the C35 protein level or mRNA level ina second biological sample).

Preferably, the C35 protein level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard C35 proteinlevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the cancer. As will beappreciated in the art, once a standard C35 protein level or mRNA levelis known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source which containsC35 protein or mRNA. Biological samples include mammalian body fluids(such as sera, plasma, urine, synovial fluid and spinal fluid) whichcontain secreted mature C35 protein, and ovarian, prostate, heart,placenta, pancreas, liver, spleen, lung, breast, bladder and umbilicaltissue which may contain precursor or mature forms of C35.

The present invention is useful for detecting cancer in mammals. Inparticular, the invention is useful during diagnosis of the followingtypes of cancers in mammals: breast, bladder, ovarian, prostate, bone,liver, lung, pancreatic, and splenic. Preferred mammals include monkeys,apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularlypreferred are humans.

Total cellular RNA can be isolated from a biological sample using thesingle-step guanidinium-thiocyanate-phenol-chloroform method describedin Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the C35 protein are then assayed using any appropriatemethod. These include Northern blot analysis (Harada et al., Cell63:303-312 (1990)), S1 nuclease mapping (Fujita et al., Cell 49:357-367(1987)), the polymerase chain reaction (PCR), reverse transcription incombination with the polymerase chain reaction (RT-PCR) (Makino et al.,Technique 2:295-301 (1990)), and reverse transcription in combinationwith the ligase chain reaction (RT-LCR).

Assaying C35 protein levels in biological sample can occur usingantibody-based techniques. For example, C35 protein expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)).

Other antibody-based methods useful for detecting C35 protein expressioninclude immunoassays, such as enzyme linked immunosorbent assay (ELISA),ELISPOT, and the radioimmunoassay (RIA).

Suitable labels are known in the art and include enzyme labels, such as,Glucose oxidase, and radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon(¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium(^(99m)Tc), and fluorescent labels, such as fluorescein and rhodamine,and biotin.

C35-specific T cells may be detected in a variety of proliferation andlymphokine secretion assays following activation by C35 presented byantigen presenting cells according to methods known in the art.Tetrameric complexes of a C35 peptide epitope bound to soluble MHCmolecules can be employed to directly stain and enumerate C35-specific Tcells in a population of cells (Lee, P. P. et al., Nature Medicine5:677-85 (1999) the entire contents of which is hereby incorporated byreference.

In addition to assaying secreted protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, ¹³¹I, ¹¹²In, ⁹⁹mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Fusion Proteins

Any C35 polypeptide can be used to generate fusion proteins. Forexample, the C35 polypeptide, when fused to a second protein, can beused as an antigenic tag. Antibodies raised against the C35 polypeptidecan be used to indirectly detect the second protein by binding to theC35. Moreover, because secreted proteins target cellular locations basedon trafficking signals, the C35 polypeptides can be used as a targetingmolecule once fused to other proteins.

Examples of domains that can be fused to C35 polypeptides include notonly heterologous signal sequences, but also other heterologousfunctional regions. The fusion does not necessarily need to be direct,but may occur through linker sequences.

In certain preferred embodiments, C35 fusion polypeptides may beconstructed which include additional N-terminal and/or C-terminal aminoacid residues. In particular, any N-terminally or C-terminally deletedC35 polypeptide disclosed herein may be altered by inclusion ofadditional amino acid residues at the N-terminus to produce a C35 fusionpolypeptide. In addition, C35 fusion polypeptides are contemplated whichinclude additional N-terminal and/or C-terminal amino acid residuesfused to a C35 polypeptide comprising any combination of N- andC-terminal deletions set forth above.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the C35 polypeptide. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the C35 polypeptide to improve stability andpersistence during purification from the host cell or subsequenthandling and storage. Also, peptide moieties may be added to the C35polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the C35 polypeptide. The addition ofpeptide moieties to facilitate handling of polypeptides are familiar androutine techniques in the art.

Moreover, C35 polypeptides, including fragments, and specificallyepitopes, can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. One reported example describes chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86(1988).) Fusion proteins having disulfide-linked dimeric structures (dueto the IgG) can also be more efficient in binding and neutralizing othermolecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the C35 polypeptides can be fused to marker sequences, such asa peptide which facilitates purification of C35. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984).)

Thus, any of these above fusions can be engineered using the C35polynucleotides or the C35 polypeptides.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing the C35polynucleotide, host cells, and the production of C35 polypeptides byrecombinant techniques. The vector may be, for example, a phage,plasmid, viral, or retroviral vector. Retroviral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.

C35 polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The C35 polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination, and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the transcripts expressed by the constructs willpreferably include a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pHE-4 (and variantsthereof); pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.;pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3,pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Amongpreferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; and pSVK3, pBPV, pMSG and pSVL available fromPharmacia. Other suitable vectors will be readily apparent to theskilled artisan. Preferred vectors are poxvirus vectors, particularlyvaccinia virus vectors such as those described in U.S. patentapplication Ser. No. 08/935,377, the entire contents of which areincorporated herein by reference.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

C35 polypeptides can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification.

C35 polypeptides can also be recovered from: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect, and mammalian cells. Depending upon thehost employed in a recombinant production procedure, the C35polypeptides may be glycosylated or may be non-glycosylated. Inaddition, C35 polypeptides may also include an initial modifiedmethionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g. C35 coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with C35 polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous C35 polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous C35 polynucleotide sequences via homologous recombination(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication No. WO 96/29411, published Sep. 26, 1996; InternationalPublication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al.,Nature 342:435-438 (1989), the disclosures of each of which areincorporated by reference in their entireties).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Differential Expression of C35 in Human BreastCarcinoma

The present inventors have characterized a full-length cDNA representinga gene, C35, that is differentially expressed in human breast andbladder cancer (FIG. 1). A 348 base pair DNA fragment of C35 wasinitially isolated by subtractive hybridization of poly-A RNA from tumorand normal mammary epithelial cell lines derived from the same patientwith primary infiltrating intraductal mammary carcinoma. (Band, V. etal., Cancer Res. 50:7351-7357 (1990). Employing primers based on thissequence and that of an overlapping EST sequence (Accession No. W57569),a cDNA that includes the full-length C35 coding sequence was thenamplified and cloned from the SKBR3 breast tumor cell line (ATCC,HTB-19). This C35 cDNA includes, in addition to the 348 bp codingsequence, 167 bp of 3′ untranslated region.

Differential expression of the C35 sequence is demonstrated in FIG. 2panel A which compares expression levels of clone C35 in poly-A RNA fromcell lines derived from normal mammary epithelium, from two primarybreast tumor nodules, and from two metastatic lung tumor nodulesisolated approximately one year later from the same patient (Band, V. etal., Cancer Res. 50:7351-7357 (1990)). Quantitative analysis indicatesthat the sequence is expressed at a more than 10 fold higher level intumor cells than in normal mammary epithelium. Low expression levels ina panel of other normal tissues is demonstrated by the Northernhybridization results of FIG. 2 panel B. Even though three times as muchpoly-A RNA was loaded from normal tissues as from the tumor cell lines,little or no expression of RNA homologous to C35 was detected after acomparable 15 hour exposure. Only after an extended 96 hour exposure waslow level expression of some homologous sequences detected in normalspleen and kidney tissues. Analysis of expression of C35 homologoussequences in poly-A RNA from three primary infiltrating ductal breastcarcinoma from different patients as well as a sample of normal breastepithelium is shown in FIG. 2 panel C. In comparison to normal breastepithelium, sequences homologous to C35 are overexpressed as much as 45and 25 fold in two of the three primary breast tumors.

The present inventors previously conducted an analysis of animmunoprotective tumor antigen expressed in several independentlyderived murine tumors and, at much reduced levels, in normal mousetissues. (See U.S. patent application filed Mar. 28, 2000, titled“Methods of Producing a Library and Methods of Directly Selecting CellsExpressing Inserts of Interest,” the entire contents of which are herebyincorporated herein by reference). In this case, a factor of 9difference between expression levels in tumor and normal tissues wasassociated with induction of an immunoprotective tumor-specificresponse. As discussed above, the expression level of C35 in some humanbreast cancers relative to normal tissue exceeds a factor of 9,suggesting that C35 might also be immunoprotective against breast cancerin these individuals.

Example 2 C35 Specific CTL are Cytolytic for C35 Positive Breast TumorCells

Although a gene product may be overexpressed in tumor cells, as is thecase for C35, it is immunologically relevant only if peptides derivedfrom that gene product can be processed and presented in associationwith MHC molecules of the tumor cells. It is conceivable that for anygiven gene product either no peptides are produced during the cellulardegradation process that satisfy the requirements for binding to the MHCmolecules expressed by that tumor, or, even if such peptides aregenerated, that defects in transport or competition for MHC molecules byother tumor peptides would preclude presentation of any peptides fromthat specific gene product. Even if relevant tumor peptides areprocessed and presented in association with human MHC in the tumorcells, it must in all cases be determined whether human T cells reactiveto these peptides are well-represented in the repertoire or whether Tcells may have been rendered tolerant, perhaps due to expression of thesame or a related antigen in some other non-homologous normal tissue.For both these reasons, therefore, it is essential to confirm thatMHC-restricted, human tumor antigen-specific T cells can be induced byC35 and that they are indeed crossreactive on human tumor cells.Relevant information on this point can be obtained through in vitrostimulation of human T cell responses with recombinant C35 or C35peptides presented by autologous antigen presenting cells.

A major technical problem in evaluating T cell responses to recombinantgene products is that a strong immune response against the expressionvector can block or obscure the recombinant specific response. This isparticularly a problem with primary responses that may require multiplecycles of in vitro stimulation. To minimize vector specific responses,it is possible to alternate stimulation by antigen presenting cellsinfected with different viral vectors recombinant for the same geneproduct. Convenient vectors include: retroviruses, adenovirus, and poxviruses.

Human PBMC were purified using Ficoll-Paque and subject to rosettingwith neuraminidase-treated sheep erythrocytes to isolate monocytes(erythrocyte rosette negative, ER⁻) and T lymphocytes (ER⁺). Dendriticcells were generated from the ER⁻ fraction by culture for 7 days inrhGM-CSF (1000 U/ml) and rhIL-4 (1000 U/ml) with fresh medium andcytokines being added every other day. At day 7, immature dendriticcells were transduced with retrovirus expressing human C35 in thepresence of polybrene (1 μg/ml) for 6 hours. Cells were washed andincubated under maturation conditions for 4 days in the presence of12.5% monocyte conditioned medium, 1000 U/ml rhGM-CSF and 1000 rhU/mlIL-4 and 1% autologous serum. At this point, the dendritic cells wereincubated with autologous T lymphocytes (cryopreserved ER fraction) at aratio of 1 DC:50 T cells for 14 days. Viable T cells were restimulatedwith autologous, irradiated EBV-B B cells infected at a multiplicity ofinfection of 1 overnight (16 hours) with a vaccinia recombinantexpressing human C35 in the presence of cytokines IL-2 (20 U/ml), IL-12(20 U/ml) and IL-18 (10 ng/ml). Cells were restimulated two more timeswith autologous EBV-B cells infected with C35-bearing retrovirus in thepresence of IL-2 and IL-7 (10 ng/ml). Cytotoxic activity was measuredafter a total of 4 stimulations by ⁵¹Cr release assay using 5000targets/well in a 4 hour assay. The results shown in Table 8 belowdemonstrate specific cytotoxic activity of C35 stimulated T cellsagainst 21NT breast tumor cells that express relatively elevated levelsof C35 but not against MDA-MB-231 tumor cells that express the same lowlevels of C35 as normal nontransformed epithelial cells.

TABLE 8 C35-specific CTL are Cytolytic for C35 Positive Breast TumorCells Target HLA Haploype E:T Cells (Effectors: 20:1 10:1 Autologous A2,A11; B8, B35) (% specific lysis) EBV-B A2, A11; B8, B35 2 1 MDA-MB-231A2; B8 3 1 C35 low (1x) 21NT A26, A31; B35, B38 22 10 C35 high (12x)K562 2 0

Example 3 C35 Expression on the Membrane of Breast Carcinoma Cells

To determine whether the C35 polypeptide product is expressed on thesurface of tumor cells, a C35 specific antiserum was prepared. BALB/cmice were immunized with syngeneic Line 1 mouse tumor cells that hadbeen transduced with retrovirus encoding human C35. Mice were bledfollowing a series of two or more immunizations. The immune sera wereemployed to detect surface expression of C35 protein by flow cytometryon three breast tumor cell lines representing high (21NT), intermediate(SKBR3), and low (MDA-MB-231 levels of expressionof the C35 transcriptin Northern blots (see FIG. 4). 1×10⁵ breast tumor cells were stainedwith 3.5 microliters of C35 specific antiserum or control, pre-bleedBALB/c serum. After a 30 minute incubation, cells were washed twice withstaining buffer (PAB) and incubated with FITC-goat anti-mouse IgG (1μg/sample) for 30 minutes. Samples were washed and analyzed on an EPICSElite flow cytometer. The results presented in FIG. 4 demonstratemembrane expression of the C35 antigen recognized by the specific immuneserum at high levels on tumor line 21NT (panel A), intermediate levelsfor tumor line SKBR3 (panel B), and undetectable levels in tumor lineMDA-MB-231 (panel C). The high level of reactivity of antibody tomembranes of tumor cells that express elevated levels of C35 transcriptssuggests that C35 specific antibodies may serve as effectiveimmunotherapeutic agents for the large number of breast carcinoma thatoverexpress this gene product (see FIGS. 2 and 3).

Example 4 A Deregulated Ribosomal Protein L3 Gene Encodes a SharedMurine Tumor Rejection Antigen

The present inventors have developed novel antigen discovery technologythat allows for the selection of genes encoding CTL epitopes from a cDNAlibrary constructed in a poxvirus. Using this technology the presentinventors have determined that a shared murine tumor antigen is encodedby an alternate allele of the ribosomal protein L3 gene. The immunogenicL3 gene is expressed at significant albeit reduced levels in normaltissues including thymus. Immunization with a vaccinia recombinant ofthe immunogenic L3 cDNA induces protective immunity against tumorchallenge. It is of particular interest that a deregulated allele of ahousekeeping gene can serve as an immunoprotective antigen and thatthymic expression does not preclude immunogenicity of an upregulatedtumor product. These observations emphasize that tolerance to aself-protein is not absolute but must be defined in relation toquantitative levels of expression. The ribosomal protein described maybe representative of a class of shared tumor antigens that arise as aresult of deregulated expression of a self-protein without compromisingimmune tolerance to normal tissues. Such antigens would be suitable forimmunotherapy of cancer in vital organs.

Methods

Total RNA was isolated from BCA 39 tumor cells using the Perfect RNATotal RNA Isolation Kit (5 Prime 3 Prime, Inc., Boulder, Colo.). Poly A+mRNA was isolated from the total RNA using Dynabeads (Dynal, LakeSuccess, N.Y.). Two micrograms of poly A+ mRNA was converted to doublestranded cDNA using the Great Lengths cDNA Synthesis Kit (Clontech, PaloAlto, Calif.). The double stranded cDNA was then inserted in vacciniavirus vector v7.5/tk.

Balb/cByJ (Jackson Labs) mice were immunized intraperitoneally with2×10⁶ irradiated (6,500 cGy) BCA 34 cells. Two weeks later the mice wereboosted by subcutaneous injection of 2×10⁶ irradiated BCA 34 cells. Oneweek following the second immunization splenocytes were harvested,divided into 12 parts and cultured in 12 well plates with 6×10⁵irradiated (10,000 cGy), mitomycin C treated BCA 34 cells per well. Atweekly intervals viable T cells were purified using Lympholyte-M(Accurate Chemical, Westbury, N.Y.) and cultured in 12 well plates at1.5×10⁶ T cells per well. To each well was also added 4×10⁶ irradiated(5000 cGy) Balb/c spleen, along with 6×10⁵ irradiated, mitomycin Ctreated BCA 34 cells.

A specific vaccinia recombinant that encodes the well characterizedovalbumin 257-264 peptide (SIINFEKL) that is immunodominant inassociation with H-2K^(b) was diluted with non-recombinant virus so thatit initially constituted either 0.2%, 0.01%, or 0.001% of total viralpfu. An adherent monolayer of MC57G cells (H-2^(b)) were infected withthis viral mix at m.o.i.=1 (approximately 5×10⁵ cells/well). Following12 hours infection, ovalbumin peptide-specific CTL, derived by repeatedin vitro stimulation of ovalbumin primed splenic T cells with theimmunodominant SIINFEKL peptide, were added. During this incubationthose adherent cells which were infected with a recombinant particlethat expresses the ovalbumin peptide are targeted by specific cytotoxicT cells and undergo a lytic event which causes them to be released fromthe monolayer. Following incubation with CTL, the monolayer is gentlywashed, and both floating cells and the remaining adherent cells areseparately harvested. Virus extracted from each cell population wastitred for the frequency of recombinant (BRdU resistant) viral pfu.Virus extracted from floating cells was then used as input to anotherenrichment cycle with fresh adherent MC57G cells and ovalbuminpeptide-specific CTL. It was observed that following enrichment of VVovato greater than 10% of total virus, further enrichment of therecombinant virus was accelerated if the m.o.i. in succeeding cycles wasreduced from 1 to 0.1.

Confluent monolayers of BCN in wells of a 12 well plate were infectedwith moi=1.0 vaccinia BCA39 cDNA library. At 12 hours post-infection themonolayers were washed 3× with media, and 2.5×10⁶ CTL were added to thewells in a 250 μl volume. The T cells and targets were incubated at 37°C. for 4 hours. Following the incubation the supernatant was harvested,and the monolayer gently washed 3× with 250 μl media. Virus was releasedfrom the cells by freeze/thaw, and titers determined by plaque assay onBSC1 cells. The selected virus population (floating cells in culturesthat received specific T cells) was amplified on BSC1 cells in one wellof a 12 well plate for 2 days. The virus was then harvested and titered.This viral stock was subjected to three additional enrichment cycles.The selected virus population was not amplified prior to the next cycle.

Virus from the fourth enrichment cycle was divided into 40 pools of 5pfu each. Each pool was amplified on BSC1 cells in a 96 well plate, with1 pool/well. After 4 days the virus was harvested (P1), and used toinfect monolayers of BCN in a 96 well plate at moi=5, with 1 pool perwell. As a control, a monolayer of BCN was infected with moi=5 vNotI/tk(Merschlinsky et al., Virology 190:522 (1992)). At 5 hourspost-infection, 2×10⁴ washed CTL were added to each well. The finalvolume in each well was 225 μl. The cells were incubated at 37° C. for18 hours. The cells were then pelleted by centrifugation, 150 μlsupernatant was harvested and tested for IFNg by ELISA. Twenty seven ofthe forty pools of 5 pfu were positive for the ability to stimulate CTL.Suggesting, by Poisson analysis, that specific recombinants wereenriched to greater than 20%. Individual clones were picked from 5positive pools and assayed as above.

Monolayers of B/C.N in a 6 well plate were infected with moi=1.0 ofv7.5/tk, vF5.8, or vH2.16. At 14 hours post-infection cells wereharvested along with the control targets: B/C.N, BCA 34, and BCA 39. Thetarget cells were labeled with 100 microcuries ⁵¹Chromium (Dupont,Boston, Mass.) for 1 hour at 37° C., and 10⁴ cells were added to wellsof a 96 well round bottom plate in quadruplicate. Tumor specific CTLwere added to target cells at the indicated ratios. Cells were incubatedat 37° C. for 4 hours. Supernatants were harvested and ⁵¹Cr releasedetermined. Spontaneous release was derived by incubating target cellswith media alone. Maximal release was determined by incubating targetcells with 5% Triton X 100. Percentage of specific lysis was calculatedusing the formula: % specific lysis=((experimental release−spontaneousrelease)/(maximal release−spontaneous release))×100. In each case themean of quadruplicate wells was used in the above formula.

Two micrograms of total RNA was converted to cDNA using a dT primer andSuperscript II Reverse Transcriptase (BRL, Gaithersburg, Md.). cDNA wasused as the template for a PCR using L3 specific primers; L3.Fl.S(CGGCGAGATGTCTCACAGGA) and L3.Fl.AS (ACCCCACCATCTGCACAAAG); and KlentaqDNA Polymerase Mix (Clontech) in a 20 microliter final volume. Reactionconditions included an initial denaturation step of 94° C. for 3minutes, followed by 30 cycles of: 94° C. 30 seconds, 60° C. for 30seconds, 68° C. for 2 minutes. These PCR products contained the regionof L3 between position 3 and 1252. The PCR products were purified usingCentricon 100 columns (Amicon, Beverly, Mass.), digested with Sau3AI,and resolved on a 3% Agarose/ethidium bromide gel.

Adult female Balb/cByJ mice (2 mice per group) were immunized bysubcutaneous injection of 5×10⁶ pfu of vH2.16, or v7.5/tk. Seven daysfollowing the immunization splenocytes were harvested and cultured in 12well plates along with 1 micromolar peptide L3₄₈₋₅₆(I54). After sevendays the viable T cells were purified using Lympholyte-M, and 1×10⁶ Tcells were added to wells of a 12 well plate along with 1 micromolarpeptide and 4×10⁶ irradiated (5000 cGy) Balb/c spleen cells per well.

Adult female Balb/cByJ mice were immunized by subcutaneous injection of10×10⁶ pfu of vH2.16, vPKIa, v7.5/tk or Phosphate Buffered Saline.Secondary immunizations were given 21 days later. Mice were challengedwith tumor by subcutaneous injection of 2×10⁵ BCA 34 cells twenty one(primary immunization only) or fourteen days following immunization.

Results and Discussion

Prospects for development of broadly effective tumor vaccines have beenadvanced by evidence that several self-proteins can be recognized astumor antigens by immune T cells (Van den Eynde et al., J. Exp. Med.173:1373 (1991); M. B. Bloom et al., J. Exp. Med. 185:453 (1997); VanDer Bruggen et al., Science 254:1643 (1991); Gaugler et al., J. Exp.Med. 179:921 (1994); Boel et al., Immunity 2:167 (1995); Van Den Eyndeet al., J. Exp. Med. 182:689 (1995); Kawakami et al., Proc. Natl. Acad.Sci. U.S.A. 91:3515 (1994); Kawakami et al., Proc. Natl. Acad. Sci.U.S.A. 91:6458 (1994); Brichard et al., J. Exp. Med. 178:489 (1993)).Such normal, nonmutated gene products may serve as common targetantigens in tumors of certain types arising in different individuals.Clinical evidence for induction of protective immunity followingvaccination with such shared tumor antigens is, currently, very limited(Marchand et al., Int. J. Cancer 80:219 (1999); Rosenberg et al., Nat.Med. 4:321 (1998); Overwijk et al., Proc. Natl. Acad. Sci. 96:2982(1999); Brandle et al., Eur. J. Immunol. 28:4010 (1998)). It is,moreover, not at all clear whether the T cell responses to theseself-proteins represent a surprising breakdown in immunologicaltolerance or are a consequence of qualitative or quantitative changes inthe expression of the self-proteins in tumor cells. In the latter case,normal tissue tolerance could be maintained and vaccine induced immunityto self-proteins whose expression is systematically altered in tumorsmight be applicable even to cancer of vital organs.

The present inventors have shown that a ribosomal protein allele that issystematically deregulated in multiple murine tumors during thetransformation process is a tumor rejection antigen and that theprincipal correlate of immunogenicity is a dramatic change inquantitative expression in tumors relative to normal tissues and thymus.

Previously, the present inventors have reported that cross-protectiveimmunity is induced among three independently derived murine tumor celllines (Sahasrabudhe et al., J. Immunology 151:6302 (1993)). Thesetumors, BCA 22, BCA 34, and BCA 39 were derived by in vitro mutagenesisof independent subcultures of the B/C.N line, a cloned, immortalized,anchorage-dependent, contact inhibited, nontumorigenic fibroblast cellline derived from a Balb/c embryo (Collins et al., Nature 299:169(1982); Lin et al., JNCI 74:1025 (1985)). Strikingly, immunization withany of these tumor cell lines, but not with B/C.N provided protectionagainst challenge with not only homologous tumor cells, but also againstchallenge with the heterologous tumor cell lines. Following immunizationwith any of these three tumor cell lines, CD8+ cytolytic T lymphocyte(CTL) lines and clones could be generated which in vitro displayedcrossreactive specificity for the same three tumors, but not for thenon-tumorigenic B/C.N cells from which they derived.

In order to move from an immunological definition to a moleculardefinition of this shared tumor antigen(s), the present inventorsdeveloped a novel and efficient method for the identification of genesthat encode CTL target epitopes. In this approach a cDNA library fromthe BCA 39 tumor cell line was constructed in a modified vaccinia virusexpression vector (Merchlinsky et al., Virology 238:444 (1997); E. Smithet al., Manuscript in preparation). Five hundred thousand plaque formingunits (pfu) of this library were used to infect a monolayer ofantigen-negative B/C.N cells at a multiplicity of infection (moi) of 1.Following 12 hours infection, BCA 34 tumor specific CTL were added tothe target cell monolayer at an effector to target ratio that givesapproximately 50% lysis in a standard ⁵¹Cr release assay. CTL specificfor the heterologous BCA 34 tumor cell line were used in order tofacilitate the identification of antigen(s) which are shared betweenthese two tumor cell lines. Since adherence is an energy dependentprocess, it was expected that cells that undergo a CTL mediated lyticevent would come off of the monolayer and could be recovered in thesupernatant. By harvesting virus from floating cells following cellmediated lymphocytotoxicity (CML), it was possible to enrich for viralrecombinants that had sensitized the host cell to lysis. An essentialfeature of this procedure is that it lends itself to repetition. Thevirus harvested following one cycle of enrichment can be used as inputfor additional cycles of selection using fresh monolayers and fresh CTLuntil the desired level of enrichment has been achieved. In a modelexperiment with CTL specific for a known recombinant, it was possible todemonstrate that specific recombinants could be enriched from an initialdilution of 0.001% to approximately 20% in 6 cycles of selection (Table9). At this level it is a simple matter to pick individual plaques forfurther characterization.

TABLE 9 Multiple Cycles of Enrichment for VVova A vaccinia cocktailcomposed of wild type vNotl/tk (tk+) spiked with the indicatedconcentrations of VVova (tk−) was subjected to CML Selection (12)Enrichment % VVova in Floating Cells Cycle # Expt. 1 Expt. 2 Expt. 3 moi= 1 0 0.2 0.01 0.001 1 2.1 0.3 nd 2 4.7 1.1 nd 3 9.1 4.9 nd 4 14.3 17.91.4 5 24.6 3.3 6 18.6 Moi = 0.1 5 48.8 39.3 % VVova = (Titer withBudR/Titer without BudR) × 100 nd = not determined

The poxvirus expression library was subjected to 4 cycles of selectionwith tumor-specific CTL. Individual plaques of the selected viralrecombinants were expanded and used to infect separate cultures of B/C.Ncells. These cells were assayed for the ability to stimulate specificCTL to secrete interferon gamma (IFN-gamma) (FIG. 5A), or forsensitization to lysis by the tumor-specific CTL (FIG. 5B). Ten viralclones were isolated, all of which conferred upon B/C.N the ability tostimulate a line of tumor-specific CTL to secrete IFNγ. All 10 clonescontained the same sized (1,300 bp) insert (Smith et al., unpublisheddata). Sequence analysis confirmed that clones F5.8 and H2.16 containedthe same full-length cDNA. It appeared, therefore, that all ten cloneswere recombinant for the same cDNA. In all, 6 of 6 CTL lines that weregenerated by immunization with BCA 34 demonstrated specificity for thisantigen.

A search of GenBank revealed that this cDNA is highly homologous to themurine ribosomal protein L3 gene (Peckham et al., Genes and Development3:2062 (1989)). Sequencing the entire H2.16 clone revealed only a singlenucleotide substitution that coded for an amino acid change whencompared to the published L3 gene sequence. This C170T substitutiongenerates a Threonine to Isoleucine substitution at amino acid position54. The F5.8 clone also contained this nucleotide substitution.

Since CTL recognize antigen as peptide presented by a MajorHistocompatibility Complex (MHC) molecule, it was of interest toidentify the peptide epitope recognized by these class I MHC-restrictedtumor-specific CD8+ T cells. It was considered likely that the alteredamino acid (Ile 54) would be included in the peptide recognized by theCTL. This hypothesis was supported by the demonstration that a vacciniavirus clone recombinant for only the first 199 bp (63 amino acids) ofH2.16 (vH2₁₉₉) was able to sensitize B/C.N to lysis by tumor-specificCTL (Smith et al., unpublished data). A Computer screen ofpeptide-binding motifs suggested that there are two epitopes encodedwithin this region that could associate with high affinity to the classI MHC molecule Kd (FIG. 12) (Parker et al., J. Immunology 152:163(1994)). These two peptides, L3₄₅₋₅₄ (I54) and L3₄₈₋₅₆ (I54) weresynthesized and tested for the ability to sensitize B/C.N cells to lysisby tumor-specific CTL. As shown in FIG. 7A, peptide L3₄₈₋₅₆ (I54)sensitized B/C.N to lysis, while L3₄₅₋₅₄ (I54), and the wild typeL3₄₈₋₅₆ (T54) did not. It was determined that 10 nM L3₄₈₋₅₆ (I54) wassufficient to sensitize targets to lysis by CTL, whereas 100 mM L3₄₈₋₅₆(T54) did not (FIG. 7B). These results demonstrate that peptide L3₄₈₋₅₆(I54) is a target epitope recognized by the tumor-specific CTL.

To analyze expression of the different L3 gene products, oligo-dT primedcDNA was synthesized from RNA of tumors and the B/C.N cell line fromwhich they derived. The first strand cDNA was subjected to PCRamplification using a pair of primers which amplify nearly the entiremouse L3 mRNA. Sequence analysis of these PCR products showed that B/C.Nand BCB13 L3 cDNA contained a C at position 170 (same as publishedsequence). BCB13 is a tumor cell line that was derived from the B/C.Ncell line, but that is not immunologically cross-protective with the BCAtumor cell lines (Sahasrabudhe et al., J. Immunology 151:6302 (1993)).Sequence analysis of the PCR products from the crossreactive BCA 39, BCA34, and BCA 22 tumors suggested that these cell lines express twodifferent species of L3 mRNA. One species contains a C at 170, and theother contains a T at 170, as in the H2.16 clone. The sequence of all L3cDNAs were identical except for this one base substitution.

There are two possible ways to account for the origin of the new L3 RNAin tumor cells. Either the L3 (C 170T) gene expressed in these tumors isa somatic mutant of the wild type gene or there are multiple germ linealleles of L3, at least one of which gives rise to an immunogenicproduct when deregulated during the process of tumor transformation. Weconsidered the first hypothesis unlikely because the crossreactive BCA39, BCA 34, and BCA 22 tumors were independently derived. It would beremarkable if the same mutant epitope was generated in all three tumors.On the other hand, Southern blots of different restriction digests ofgenomic DNA from BCA 39 and B/C.N suggested that there are multiplecopies of the L3 gene in the mouse genome (Smith et al., unpublisheddata). The L3 gene has also been reported to be multi-allelic in boththe rat and the cow (Kuwano et al., Biochemical and Biophysical ResearchCommunications 187:58 (1992); Simonic et al., Biochemica et BiophysicaActa 1219:706 (1994)). Further analysis was required to test thehypothesis that different L3 alleles in the germ line are subject todifferential regulation in tumors and normal cells.

The nucleotide sequence of the published L3 from position 168 to 171 isGACC. The sequence of H2.16 in this same region is GATC (FIG. 8A). Thisnew palindrome is the recognition sequence for a number of restrictionendonucleases, including Sau3AI. As shown in the restriction map of FIG.8A, a Sau3A I digest of L3 is expected to generate fragments of 200,355, 348, 289, and 84 base pairs, while a Sau 3A I digest of H2.16 wouldgenerate a 168 bp fragment in place of the 200 bp fragment. Thisdifference in the Sau 3AI digestion products was used to confirm thatthe three BCA cell lines express at least two different L3 alleles. TheL3 RT-PCR products from all 5 cell lines and thymus RNA were digestedwith Sau 3AI and analyzed on an agarose gel. As shown in FIG. 8B all 3BCA lines express both versions of L3. Remarkably, when this assay wasrepeated using greater amounts of starting material, the 168 bp fragmentwas also detectable in the digests of B/C.N, BCB13 and normal thymuscDNA (Smith et al., unpublished data). To enhance the sensitivity ofthis assay, the PCR was repeated using a P³² end-labeled 5′ L3 specificprimer. The radiolabeled PCR products were digested with Sau3AI andresolved on an agarose gel. As shown in FIG. 8C, B/C.N, BCB13 and thymuscontain the 168 bp fragment. Quantitative analysis indicates that theratio of 200 bp:168 bp fragments in the BCA tumors is 2:1 while theratio of the same fragments detected in B/C.N, BCB13, and thymus isapproximately 20:1. Low levels of expression of this immunogenic L3allele was also observed when RNA from kidney, heart, and skeletalmuscle was analyzed (Smith et al., unpublished data). These resultssuggest that gene deregulation associated with the transformationprocess in the crossreactive tumors leads to the expression of higherlevels of this germ line L3 (C170T) allele, and that this altered L3gene was not generated by somatic mutation of the L3 gene that ispredominantly expressed in normal tissues. The present inventors havetermed this new L3 allele (C170T), the immunogenic L3 allele (iL3).

It is particularly intriguing that the immunogenic L3 allele is alsoexpressed, albeit at a 10 fold reduced level, in normal thymus. Thislevel of expression is evidently not sufficient to tolerize all T cellswith functional avidity for the level of deregulated iL3 expressed insome tumors. The observation that although B/C.N and BCB13 express lowlevels of iL3, they are not susceptible to lysis by the tumor specificCTL suggests, however, that higher affinity T cells have been tolerized.This appears to be the first instance in which a tumor antigen has beenreported to be expressed in the thymus. These observations emphasizethat tolerance to a self-protein is not absolute but must be defined inrelation to quantitative levels of expression (Targoni et al., J. Exp.Med. 187:2055 (1998); C. J. Harrington et al., Immunity 8:571 (1998)).

If broadly effective vaccines are to be developed based on expression ofshared tumor antigens, then it is critical to demonstrate that suchantigens can be immunoprotective. The largest number of shared antigenshave been identified for human tumors, but clinical Immunotherapy trialsemploying these antigens have so far been inconclusive, in part becauseof uncertainty regarding optimal vaccination strategies (Pardoll, D. M.,Nat. Med. 4:525 (1998)). In mice, where immunotherapeutic strategiescould be more thoroughly investigated, very few shared tumor antigenshave been identified. It was, therefore, of considerable interest todetermine whether immunization with iL3 recombinant vaccinia virus wouldinduce tumor specific CTL and protect mice from tumor challenge(Overwijk et al., Proc. Natl. Acad. Sci. 96:2982 (1999); Moss, B.,Science 252:1662 (1991); Irvine et al., J. Immunology 154:4651 (1995);McCabe et al., Cancer Research 55:1741 (1995); Estin et al., Proc. Natl.Acad. Sci. 85:1052 (1988); J. Kantor et al., JNCI 84:1084 (1992); V.Bronte et al., Proc. Natl. Acad. Sci. 94:3183 (1997)). Immunization ofBalb/c mice with vaccinia virus recombinant for the iL3 gene (H2.16)generated CTL that were able to lyse both BCA 34 and BCA 39 tumor cells,but not B/C.N in vitro (FIG. 9A). Mice immunized twice or even once withvaccinia virus recombinant for iL3 were able to reject challenge withBCA 34 tumor cells (FIGS. 9B and 9C). Mice immunized with empty viralvector, or control vaccinia recombinant for the Inhibitor Protein ofcAMP-dependent Protein Kinase (PKIa) were unable to reject this tumorchallenge (Olsen, S. R. and Uhler, M. D., J. Biol. Chem. 266:11158(1991); Mueller et al., Manuscript in Preparation). These resultsdemonstrate that the iL3 self-protein is an immunoprotective tumorantigen.

The present inventors have developed a new strategy to identify genesthat encode CTL epitopes based on CTL-mediated selection from a tumorcDNA library in a modified vaccinia virus vector (Merchlinsky et al.,Virology 238:444 (1997); E. Smith et al., manuscript in preparation). Wehave applied this strategy to identify a deregulated housekeeping genethat encodes a tumor rejection antigen shared by three independentlyderived murine tumors. This ribosomal protein may be representative of alarger class of immunoprotective shared tumor antigens that becomeimmunogenic as a result of deregulated expression of self-proteinswithout compromising immune tolerance to normal tissues. Such antigenswould be well suited for immunotherapy of cancer in vital organs.

Example 5 Expression and Immunogenicity of C35 Tumor Antigen

RNA transcripts of the novel C35 tumor gene are overexpressed in 70%(12/17) of primary human breast carcinomas examined and 50% (5/10) ofbladder carcinomas examined when compared to expression in normal humantissues. The full-length gene encodes a novel 115 amino acid protein ofunknown function. A monoclonal antibody, 2C3, has been selected thatstains the surface membrane of cells expressing C35 by flow cytometricanalysis. In addition, human cytotoxic T lymphocytes (CTL) have beengenerated in vitro that specifically lyse C35+ breast and bladdertumors. The ability to generate C35-specific CTL in vitro from normalhuman donors suggests the absence of tolerance to the overexpressedprotein. Overexpression of C35 in tumors of different individuals andthe ability to induce humoral and cellular immune responses make C35apromising candidate for immunotherapy.

Material and Methods

Cell lines: Human mammary carcinoma cell lines BT20, BT474, MCF7,MDA-MB231, SKBR3, T47D (supplied by ATCC) were grown in RPMI-1640(BioWhitaker, Walkersville, Md.) supplemented with 10% fetal bovineserum (Biofluids, Rockville, Md.). An immortalized line derived fromnormal breast epithelium, H16N2, two metastastic tumors, 21-MT1 and21-MT2, and two primary tumors, 21-NT and 21-PT all derived from thesame patient, and grown in DFCI medium (Band, V. and Sager, R., “TumorProgression in Breast Cancer” in Neoplastic Transformation in Human CellCulture, J. S. Rhim and A. Dritschilo eds., The Human Press Inc.,Totowa, N.J. (1991), pp. 169-78) were generously provided by Dr. VimlaBand, New England-Tufts Medical Center. The bladder tumor cell lineppT11A3 was derived from the immortalized nontumorigenic cell lineSV-HUC. These bladder cell lines were generously provided by Dr.Catherine Reznikoff, University of Wisconsin Clinical Cancer Center, andgrown in F12 medium supplemented with 1% FBS, 0.025 units insulin, 1 μghydrocortisone, 5 μg transferrin, 2.7 g dextrose, 0.1 μM non-essentialamino acids, 2 mM L-glutamine, 100 units penicillin, and 100 μgstreptomycin per 500 ml. Normal proliferating breast epithelial cells(MEC) were purchased from Clonetics (BioWhittaker) and maintainedaccording to the supplier's directions.

RNA extraction and Northern Blot Analysis: Cell lines were harvested forRNA extraction at approximately 80% confluency. Cells were harvested andlysed in QG buffer from Qiagen RNAeasy kit. Total RNA was extracted asper manufacturer's protocol and stored at −80° C. as precipitates withGITC and alcohol. Tissue samples were provided by the Cooperative HumanTissue Network as snap frozen samples, which were homogenized in lysisbuffer for use in the RNAeasy protocol. For Northern blots, mRNA wasextracted from total RNA (30 μg total RNA/well) using Dynal's (LakeSuccess, N.Y.) oligo-dT₂₅ magnetic beads and electrophoresed in 0.8%SeaKemLE (FMC Bioproducts) with 3% formaldehyde. The mRNA was blottedonto Genescreen Plus (NEN) in 10×SSC overnight by capillary blot, thenbaked for 2 hours at 80° C. Membranes were probed with random-primed³²P-labeled cDNA probes (Prime-It, Stratagene, LaJolla, Calif.) at 10⁶cpm/ml Quickhyb solution (Stratagene), at 68° C. as per manufacturer'sprotocol. Blots were exposed to Xray film and/or phosphorimager screensovernight. Expression on all blots was normalized to a housekeepinggene, such as GAPDH or beta actin.

Subtractive Hybridization: PCR Select cDNA Subtraction Kit (Clontech,Palo Alto, Calif.), based on Representational Difference Analysis asfirst described by Lisitsyn et al. (Lisitsyn, N. and Wigler, N. M.,Science 259:946-51 (1993)), was employed as per manufacturer's protocolto generate cDNAs enriched for genes overexpressed in tumor compared tonormal breast cell lines. Briefly, oligo-dT-primed double stranded cDNAwas synthesized from 2 μg high quality, DNase-treated mRNA from tumorand normal cells. Adaptors were ligated to short blunt-end (Rsa1digested) tumor sequences and hybridized with excess Rsa1 digestednormal fragments. Following 32 hour hybridization, suppression PCR(Clontech) allowed preferential amplification of overexpressed tumorsequences using adaptor sequences as primers. The products of the PCRamplification were cloned into pT7Blue3 (Novagen, Madison, Wis.) togenerate a subtracted library. Clones were grown in LB/ampicillin (100μg/ml) in 96-well format, inserts were PCR amplified from the overnightcultures and PCR products were spotted on Genescreen Plus using BioDotmanifold (BioRad, Hercules, Calif.). Duplicate dot blots were thenprobed with random-primed tumor or normal cDNA, or, alternatively, thePCR products of the forward and reverse subtractive hybridizations.Clones that appeared to be overexpressed in the tumor cDNA and forwardsubtraction (tumor minus normal) were analyzed by Northern Blot (asdescribed above) to confirm differential gene expression.

cDNA library and full length gene: Oligo-dT primed double stranded cDNAwas generated from SKBR3 cell line using SMART cDNA Synthesis (ClontechLaboratories), followed by phenol:chloroform:isoamyl alcohol extraction.Primers were synthesized (C35 sense: 5′-GCGATGACGGGGGAGCC, and C35antisense: 5′-CCACGGAATCTTCTATTCTTTCT; Fisher Oligos, The Woodlands,Tex.) to amplify the coding region of C35, based on the open readingframe deduced from EST homologies, Accession # W57569, in particular.PCR products were cloned into pT7Blue 3 (Novagen).

Vaccinia and Retroviral C35 recombinants: The coding sequence of C35 wassubcloned from the library into vaccinia transfer plasmid, pVTK0 atBamHI/SalI sites in a defined orientation. Recombinant virus wasgenerated by transfection of pVTK0.C35 along with NotI and ApaI digestedV7.5/TK viral DNA into fowlpox virus infected BSC-1 cells. As describedelsewhere (U.S. Utility patent application Ser. No. 08/935,377;PCT/US98/24029; T Cells Specific for Target Antigens and Vaccines BasedThereon), this is an efficient method for construction of vaccinia virusrecombinants. The C35 gene was also cloned into a retroviral vectorpLXSN, and viral stocks were generated by co-transfection of 293-GPcells with pVSVg for pseudotyping. Supernatants including infectiousvirus were collected 48 hours later.

Generation of C35-specific 2C3 monoclonal antibody and FACS analysis:Line1 mouse small cell lung carcinoma cells were infected withC35-retrovirus, and 10³-2×10⁴ cells were injected into three BALB/cByJmice. Following 21 days, serum was harvested from retro-orbital bleedsand checked for reactivity with human tumor cells known to express low(MDA-MB-231) or very high (21NT) levels of C35 mRNA. Spleens were alsoharvested for the production of hybridomas by the fusion of spleen cellswith P3 myeloma cells using standard mouse hybridoma technology. ELISAwas used to screen HAT resistant clones for the presence of Ig. Highproducers were isotyped, quantitated, and used to screen C35+ and C35−cell lines by flow cytometry. Hybridoma clone supernatants containing 1μg IgG were incubated with 10⁶ cells in PAB (PBS, 1% BSA, 0.1% azide)for 30 min on ice, followed by 3 washes with PAB, and incubation withgoat anti-mouse IgG conjugated to FITC (Southern Biotechnology,Birmingham, Ala.) for 30 minutes on ice. One hybridoma clone, 2C3,recapitulated the surface staining seen with the immune serum (FIG. 14)and was selected for expansion and antibody purification (BioExpress,West Lebanon, N.Y.).

Generation of human C35-specific T cell line: Peripheral blood derivedfrom a healthy female donor (HLA A2, 11, B35, 44) was separated intoerythrocyte-rosette positive fraction (a source of total T lymphocytes)and negative fraction (a source of monocytes). The T lymphocytes werecryopreserved for later use while the monocytes were incubated underconditions to generate dendritic cells (DC). Maturation of DCs wasinduced as described by Bhardwaj and colleagues (Bender, A. et al., J.Immunol. Meth. 196:121-35 (1996); Reddy, A. et al., Blood 90:3640-46(1997); Engelmayer, J. et al., J. Immunology 163:6762-68 (1999)) withsome modifications. hGM-CSF and hIL-4 (1000 U/ml) were added every otherday. At day 7, non-adherent, immature DC were incubated with aretrovirus recombinant for C35 for 6 hours in the presence of GM-CSF andIL-4. At this point, the retroviral supernatant was washed out andimmature dendritic cells were subjected to maturation conditions, whichagain included GM-CSF, IL-4 as well as 12.5% monocyte conditioned medium(MCM). After 4 days, these mature, C35-expressing DC were used tostimulate autologous T cells at a ratio of 1 DC:50 T cells for a periodof 14 days. A fresh pool of autologous DC were generated forrestimulation of the T cells, but this time they were infected after 48hours of maturation in MCM with a vaccinia virus recombinant for C35.Cytokines IL-2 (20 U/ml), IL-12 (20 U/ml) and IL-18 (10 ng/ml) wereadded and a 1:50 ratio of DC:T cells was maintained. Following 12 daysculture, T cells were stimulated for 7 additional days with EBV-B cellsinfected with C35 recombinant retrovirus and with addition of IL-2 (20U/ml) and IL-7 (10 ng/ml). Cytokines were all purchased from R&D Systems(Minneapolis, Minn.). At this point, the cells were >90% CD8⁺ and weretested for activity in a standard ⁵¹Cr Release assay. Briefly, onemillion target cells were incubated with 100 uCi ⁵¹Cr, washed, thenincubated with CTL effectors for 4 hours in RPMI-1640, supplemented with10% human AB serum (BioWhittaker). Activity of the CTL is expressed asthe percent of specific lysis, measured as (⁵¹Cr released into thesupernatant upon lysis of labeled targets by CTL−spontaneousrelease)/(maximal release−spontaneous release).

Results

Characterization of C35: The sequence of clone C35, differentiallyexpressed in human breast tumor cells, is not homologous to any knowngene in Genbank, but homologous EST sequences (prototype Accession#W57569) were identified. Homologous human EST fragments are present inNCI CGAP (Cancer Genome Anatomy Project) libraries, including tumors ofbrain, lung and kidney (A# AA954696), Soares ovary (A# AA285089) andparathyroid tumors (A# W37432), an endometrial tumor (A#AA337071), andcolon carcinoma (A# AA313422). An open reading frame was identified thatencodes a 115 amino acid protein (FIG. 10A). The full-length gene wasisolated from a cDNA library of the breast adenocarcinoma cell lineSKBR3. Sequencing of full-length transcripts from the cell lines SKBR3,21MT2-D, and H16N2 confirmed that there were no point mutations in thecDNA; the transcript is 100% homologous in C35^(hi) cell lines, as wellas C35^(lo) cell lines. The C35 gene aligns on human chromosome 17q12(A# AC040933) and mouse chromosome 11 (A# AC064803). Exons were deducedfrom homologies with cDNA EST sequences, as well as GRAIL predictions.Interestingly, the gene for C35 is within 1000 base pairs of theHer2/neu oncogene and within 2000 bp of the gene for Growth FactorReceptor-Bound Protein 7 (GRB7), a tyrosine kinase that is involved inactivating the cell cycle and that is overexpressed in esophagealcarcinomas (Tanaka, S. et al., J. Clin. Invest. 102:821-27 (1998)) (FIG.10B). Her2/neu protein overexpression has been correlated with geneamplification in 30% breast tumors and is associated with poor clinicalprognosis (Slamon, D. J. et al., Science 235:177-82 (1987)).

Predicted protein motifs in the C35 amino acid sequence include: caseinkinase II phosphorylation sites at amino acids 38 to 41 (TYLE), 76 to 79(SKLE), and 97 to 100 (SNGE); an N-myristoylation site at amino acids 60to 65 (GGTGAF); and a cAMP- and cGMP-dependent protein kinasephosphorylation site at amino acids 94 to 97 (RRAS). Finally, the C35protein contains a prenylation motif at the COOH-terminus, amino acids112 to 115 (CVIL). Prenylation, the covalent attachment of a hydrophobicisoprenoid moiety, is a post-translational modification that promotesmembrane association and also appears to mediate protein-proteininteractions (Fu, H.-W. and Casey, P. J., Recent Progress in HormoneResearch 54:315-43 (1999)). Prenylation has been shown to be requiredfor localization and transforming potential of the oncogenic Ras familyproteins to the cell surface (Jackson, J. H. et al., Proc. Natl. Acad.Sci. U.S.A. 87:3042-46 (1990); Hancock, J. F. et al., Cell 57: 1167-77(1989)). Inhibitors of prenylation have been shown to possess anti-tumoractivities, such as slowing tumor growth (Garcia, A. M. et al., J. Biol.Chem. 268:18415-18 (1993)) and to promote rejection in animal models(Kohl, N. E. et al., Nature Med. 1:792-97 (1995)). Three O-glycosylationsites are predicted at or near the amino terminus—thr8, ser2, and ser9using NetOGlyc2.0.

C35 Transcript is Overexpressed in Breast and Bladder Carcinoma: Anideal target antigen for tumor immunotherapy would be abundantlyexpressed in multiple independent carcinomas, and would be absent orminimally expressed in normal proliferating and vital tissues.Differential expression of C35 was confirmed by Northern blot analysis.C35 is expressed in 7/10 human tumor cell lines at levels 10-25× higherthan expression in a normal immortalized breast epithelial cell line,H16N2 (FIG. 11A). Importantly, C35 expression is shared among linesderived from both primary (21NT, 21PT) and metastatic (21 MT1, 21MT2)lesions of a single patient, suggesting its expression may be associatedwith early events in the process of tumor transformation. In addition,the overexpression of C35 is shared among independently derived humanmammary carcinoma cell lines, including SKBR3, T47D, and BT474.Interestingly, the C35 expression pattern in SKBR3, MDA MB231, H16N2 andtumors derived from the same patient correlates with Her2/neuexpression, which may be associated with the close genomic proximity ofthe two genes and the incidence of HER2/neu gene amplification.

To investigate whether C35 expression in patient derived tumors isclinically relevant for development of a cancer vaccine, mRNA wasextracted from snap frozen human tissue samples obtained from theCooperative Human Tissue Network (CHTN). 70% of primary breast tumorsamples overexpress C35 transcript (FIG. 11B), and 35% (7/20) of thesebreast adenocarcinomas overexpress at levels 10-70 fold higher thannormal breast. Overexpression of C35 is also seen in 50% of bladdercarcinoma primary specimens examined (FIG. 12), while 20% (3/14) ofprimary bladder carcinoma express at levels greater than 10 fold higherthan normal bladder. Overexpression of C35, at levels 9× or greater, wasnot detected in panels of ovarian (0/7), prostate (0/5), or colon (0/15)carcinomas (data not shown).

2C3 Monoclonal Antibody reacts with C35+ cells: In order to confirmdifferential expression of the gene product encoded by C35, a monoclonalantibody against the shared tumor antigen was selected. Hybridomas wereproduced by immunizing mice with a poorly immunogenic BALB/cByJ tumorcell line, which had been transduced with a retroviral human C35recombinant. Hybridoma clones were screened for their ability to stainC35++ breast and bladder tumor cell lines (FIGS. 13A and 13B).Non-tumorigenic breast H16N2 and bladder SV-HUC epithelial cell linesdid not show a significant shift in fluorescence intensity when comparedto the isotype control. In contrast, 2C3 monoclonal antibodyspecifically stained C35+ breast tumors, SKBR3 and 21-NT-D, and bladdertumor ppT11A3. The staining was carried out on cells that were neitherfixed nor permeabilized, indicating that 2C3 antibody recognizes asurface molecule.

Inhibition of Tumor Growth with C35 Antibodies:

Antibodies are useful tools to detect diagnostic markers of cancer, butthey may also have potential use for therapeutic applications. HumanizedHer2/neu specific antibody (Herceptin) has been successfully employedfor treatment of some breast cancers. Herceptin binds HER2/neu anddownregulates signal transduction from the growth factor receptor.Growth inhibition studies were performed with C35-specific 2C3 antibody.21NT-D breast tumor and H16N2 “normal” breast cell lines were grown invitro in the presence of various antibody concentrations. An XTT assaywas performed to evaluate cell expansion at 72 hours. Results shown inFIG. 14 indicate that 2C3 inhibits growth of 21NT tumor cells byapproximately 50% at concentrations as low as 1 μg/ml.

A C35 Class I epitope is HLA-A2 Restricted:

Establishment of self-tolerance is a major obstacle to development ofvaccines based on self proteins. Tolerance, however, must be defined interms of quantitative levels of expression. It is possible that evenwhile high affinity antigen-specific T cells are tolerized, T cells withlower affinity receptors that do not have functional avidity for a lowconcentration of antigen escape tolerance induction. These same T cellscould, however, subsequently become functionally significant if there ismarkedly increased avidity associated with overexpression of the targetantigen. Even if they are few in number, such T cells could be expandedby the most fundamental of immunological manipulations, vaccination.

C35 is a self-protein expressed at low basal levels in normal humantissues. It was, therefore, necessary to determine if human T cells aretolerant to C35 at levels of expression characteristic of carcinomas.The only way to exclude tolerance is by demonstrating responsiveness,and the only way to demonstrate responsiveness short of a clinical trialis by in vitro stimulation. Human T cells and autologous dendritic cellswere derived from PBL from a normal donor. The T cells were primed byalternate stimulation with autologous dendritic cells infected withretroviral or pox virus recombinants of the C35 cDNA. CTL recovered invitro following several cycles of stimulation were analyzed for theirability to lyse C35+ target tumor cells (FIG. 15) or to secretecytokines in response to antigen induced activation (FIG. 16). Thetargets either endogenously expressed C35 and/or HLA-A2.1, or wereengineered to express these proteins via standard transfection with aC35-recombinant mammalian expression vector, or by infection withC35-recombinant vaccinia virus. Previous studies have demonstrated thatprotein expression by vaccinia virus is an efficient means of targetingpeptides to the MHC-I processing pathway (Moss, B., Science 252:1662-67(1991).

Following several rounds of stimulation, both a bulk T cell line and a Tcell clone were selected that differentially lyse C35+ tumor cellsrelative to C35^(lo) H16N2 normal breast epithelial cell line in a ⁵¹Crrelease assay (FIGS. 15A and B). The HLA-A2 restricted C35-specific CTLclone 10G3 efficiently lysed the HLA-A2 transfected tumorigenic cellline, 21-NT.A2, which expresses C35 antigen at levels 15× greater thanH16N2 and is stained with 2C3 monoclonal antibody. Specific lysis wasalso with the HLA-A2+ bladder tumor cell line ppT11A3 compared to thenon-tumorigenic bladder cell line SV-HUC from which it was derived (FIG.15B). The data demonstrate CTL sensitivity of tumors that express highlevels of C35 with minimal lysis of C35^(lo) nontumorigenic immortalizedcell lines. Importantly, the same CTL are not reactive with MEC, aprimary culture of non-immortalized, non-transformed, HLA-A2⁺ breastepithelial cells that do not express C35 at significant levels. Furtherevidence to support C35+ tumor recognition by the T cells is shown inFIGS. 16A and B. The T cells secrete IFN-gamma and TNF-alpha in responseto C35+, HLA-A2+ stimulator. Again, the non-tumorigenic, C35^(lo) cellline H16N2.A2 did not induce cytokine secretion by C35-specific T cells.However, infection of this line with vaccinia virus recombinant for C35confers the ability to activate the T cells. Since the T cells do notsecrete IFN-gamma or TNF-alpha in response to H16N2.A2 transduced withan irrelevant protein L3, this indicates that the response is specificto C35 protein expression (FIGS. 16A and B).

Following several rounds of stimulation, both a bulk T cell line and a Tcell clone were selected that differentially lyse C35+, HLA-A2+ tumorcells in a ⁵¹Cr release assay. The C35-specific CTL did not lyse theHLA-A2 transfected non-tumorigenic breast epithelial cell line, H16N2.A2(FIGS. 15A and B), although this cell line does express C35 at lowlevels based on the Northern blot data shown in FIG. 11A. However,C35-specific CTL efficiently lysed the HLA-A2 transfected tumorigeniccell line, 21-NT.A2, which expresses C35 antigen at levels 15× greaterthan H16N2 and is stained with 2C3 monoclonal antibody. C35⁺tumor-specific lysis was also shown with the bladder tumor cell lineppT11A3 compared to the non-tumorigenic bladder cell line SV-HUC fromwhich it was derived. The data demonstrate CTL sensitivity of tumorsthat express high levels of C35 with minimal lysis of C35^(lo)nontumorigenic immortalized cell lines. Importantly, the same CTL arenot reactive with MEC, a primary culture of non-immortalized,non-transformed, HLA-A2⁺ breast epithelial cells that do not express C35at significant levels. Further evidence to support C35+ tumorrecognition by the T cells is shown in FIGS. 16A and B. The T cellssecrete IFN-gamma and TNF-alpha in response to C35+, HLA-A2+ stimulator.Again, the non-tumorigenic, C35^(lo) cell line H16N2.A2 did not inducecytokine secretion by C35-specific T cells. However, infection of thisline with vaccinia virus recombinant for C35 confers the ability toactivate the T cells. Since the T cells do not secrete IFN-gamma orTNF-alpha in response to H16N2.A2 transduced with an irrelevant proteinL3, this indicates that the response is specific to C35 proteinexpression.

The C35-specific T cells were generated from a donor with HLA haplotypeA2, A11, B8, B35. The bladder cell lines, SV-HUC and ppT11A3 derive froma donor with haplotype HLA-A2, B18, B44. However, since the H16N2immortalized breast epithelial cell line and 21-NT and 21-MT breasttumor cell lines derived from the same HLA-A2 negative donor, these celllines had to be transfected with HLA-A2.1 to provide a required MHCrestriction element for recognition by HLA-A2 restricted 10G3 T cellclone (FIGS. 16A and B). The T cells were strongly stimulated to secretethese lymphokines by the breast lines that expressed both C35 and HLA-A2(compare 21-MT2 with 21-MT2.vvA2). The data indicate that there is atleast one HLA-A2.1 defined epitope of C35.

Deletion mutants of C35 coding region were constructed to identify cDNAsegments that encode the peptide epitope recognized by the CTL. FIGS.15A and B demonstrates almost equivalent IFN-gamma and TNF-alphasecretion by T cells stimulated with the full length C35 or a truncatedmutant encompassing only the first 50 amino acids.

Discussion

C35 is a novel tumor antigen that is overexpressed in breast and bladdercarcinoma. The gene has properties that make it a promising candidatefor tumor immunotherapy. It is expressed in a significant number oftumors derived from different individuals. Expression in vital normaltissues is relatively low, reducing the risk of autoimmune reactionsand, equally important, making it unlikely that immune cells have beenrendered tolerant to the gene product. C35 is characterized as a “tumorantigen” since C35 expressing dendritic cells induce autologous tumorspecific human cytotoxic T lymphocytes in vitro.

C35 is a novel gene product of unknown function. However, our studieswith monoclonal antibodies have provided some insight into thelocalization of the protein. Both serum and a monoclonal antibodyderived from a C35-immunized mouse specifically stain unfixed cells thatexpress C35. This suggests that the antibody recognizes a tumor surfacemembrane protein. Although the protein sequence does not conform withknown transmembrane motifs based on hydrophobicity, the existence of aprenylation site at the COOH terminus suggests insertion into themembrane. Prenylation is a post-translational lipid modification thatproduces a substantially more hydrophobic protein with high affinity forthe membrane (Fu, H.-W. and Casey, P. J., Recent Progress in HormoneResearch 54:315-43 (1999)). Other proteins that contain prenylationsites include the Ras oncogene family. Ras GTPases act in signaltransduction cascades with MAPK to induce cell division andproliferation. Ras proteins are anchored to the plasma membrane viaprenylation, but the proteins remain in the cytoplasmic face of themembrane. Therefore, it is possible that C35 also remains on thecytoplasmic side of the membrane, but there may be sufficient transportto the outer surface to be detected with a specific antibody.

C35-specific antibodies are valuable tools for studying the proteinexpression of C35, to corroborate Northern blot analysis, and for use inassays such as Western blots and immunohistochemistry. In addition,these antibodies may have therapeutic benefits, such as has beenrecently been demonstrated for Herceptin (Baselga, J. et al., J. Clin.Oncol. 14:737-44 (1996); Pegram, M. D. et al., J. Clin. Oncol.16:2659-71 (1998)), an antibody to the tumor-associated antigen HER2-neu(c-erbB-2) (Schechter, A. L. et al., Nature 312:513-16 (1984).Herceptin's anti-tumor effects include binding the epidermal growthfactor receptor, which inhibits tumor cell growth, and elicitingantibody dependent cell-mediated cytotoxicity (Dillman, R. O., CancerBiotherapy & Radiopharmaceuticals 14:5-10 (1999).

Example 6 Induction of Cytotoxic T Cells Specific for Target Antigens ofTumors

Human tumor-specific T cells have been induced in vitro by stimulationof PBL with autologous tumors or autologous antigen presenting cellspulsed with tumor lysates (van Der Bruggen, P. et al., Science 254:1643-1647 (1991); Yasumura, S. et al., Cancer Res. 53: 1461-68 (1993);Yasumura, S. et al., Int. J. Cancer 57: 297-305 (1994); Simons, J. W. etal., Cancer Res. 57: 1537-46 (1997); Jacob, L. et al., Int. J. Cancer71:325-332 (1997); Chaux, P. et al., J. Immunol. 163:2928-2936 (1999)).PBL have been derived from either patients deliberately immunized withtumor, with tumor modified to enhance its immunogenicity, or with tumorextracts, or patients whose only prior stimulation was in the naturalcourse of disease. T cells with reactivity for infectious agents couldbe similarly derived by in vitro stimulation of T cells with autologouscells that have been either infected in vitro or were infected in vivoduring the natural course of exposure to the infectious agent. CD4+ andCD8+ T cells or antibody selected under these or other conditions to bespecific for either tumor cells or cells infected with either a virus,fungus or mycobacteria or T cells or antibodies specific for the targetantigens of an autoimmune disease could be employed in the selection andscreening methods of this invention to detect and isolate cDNA thatencode these target antigens and that have been incorporated into arepresentative cDNA library.

In spite of demonstrated success in the induction of human T cellresponses in vitro against a number of antigens of tumors and infectedcells, it is not certain that these represent the full repertoire ofresponses that might be induced in vivo. Because safety considerationslimit the possibilities of experimental immunization in people, there isa need for an alternative animal model to explore immune responses tohuman disease antigens. The major obstacle to developing such a model isthat numerous molecules expressed in normal human cells are stronglyimmunogenic in other species. It is, therefore, necessary to devise ameans of inducing tolerance to normal human antigens in another speciesin order to reveal immune responses to any human disease-specificantigens. It is now recognized that activation of antigen-specific Tlymphocytes requires two signals of which one involves presentation of aspecific antigenic complex to the T cell antigen receptor and the secondis an independent costimulator signal commonly mediated by interactionof the B7 family of molecules on the surface of the antigen presentingcell with the CD28 molecule on the T cell membrane. Delivery of anantigen-specific signal in the absence of a costimulator signal not onlyfails to induce T cell immunity but results in T cell unresponsivenessto subsequent stimulation (Lenschow, D. J. et al., Ann. Rev. Immunol.14:233-258 (1996)). Additional studies have revealed a key role foranother pair of interactions between the CD40 molecule on the antigenpresenting cell and CD40 ligand on the T cell. This interaction resultsin upregulation of the B7 costimulator molecules (Roy, M. et al., Eur.J. Immunol. 25:596-603 (1995)). In the presence of anti-CD40 ligandantibody either in vivo or in vitro, the interaction with CD40 isblocked, B7 costimulator is not up regulated, and stimulation with aspecific antigenic complex results in T cell tolerance rather than Tcell immunity (Bluestone, J. A. et al., Immunol. Rev. 165:5-12 (1998)).Various protocols to block either or both CD40/CD40 ligand interactionsand B7/CD28 interactions have been shown to effectively inducetransplantation tolerance (Larsen, C. et al., Nature 381:434-438 (1996);Kirk et al., Nature Medicine 5:686-693 (1999)). An example of the effectof anti-CD40 ligand antibody (anti-CD154) in blocking the reactivity ofmurine T cells to specific transplantation antigens is shown in FIG. 17.DBA/2 (H-2^(d)) mice were immunized with 10⁷ C57B1/6 (H-2^(b)) spleencells intraperitoneally and, in addition, were injected with eithersaline or 0.5 mg monoclonal anti-CD40 ligand antibody (MR1, anti-CD154,Pharmingen 09021D) administered both at the time of immunization and twodays later. On day 10 following immunization, spleen cells from thesemice were removed and stimulated in vitro with either C57B1/6 or controlallogeneic C3H(H-2^(k)) spleen cells that had been irradiated (20 Gy).After 5 days in vitro stimulation, C57B1/6 and C3H specific cytolyticresponses were assayed at various effector:target ratios by ⁵¹Cr releaseassay from specific labeled targets, in this case, either C3H or C57B1/6dendritic cells pulsed with syngeneic spleen cell lysates. The resultsin FIG. 17 show that significant cytotoxicity was induced against thecontrol C3H alloantigens in both saline and anti-CD154 treated micewhereas a cytotoxic response to C57B1/6 was induced in the salinetreated mice but not the anti-CD154 treated mice. This demonstratesspecific tolerance induction to the antigen employed for immunestimulation at the time CD40/CD40 ligand interactions were blocked byanti-CD154.

A tolerization protocol similar to the above employing either anti-CD154 alone or a combination of anti-CD154 and anti-B7 or anti-CD28 couldbe employed to induce tolerance to normal human xenoantigens in miceprior to immunization with a human tumor. In one embodiment, the normalantigens would be expressed on immortalized normal cells derived fromthe same individual and tissue from which a tumor cell line is derived.In another embodiment, the normal and tumor antigens would derive fromcell lysates of normal and tumor tissue of the same individual eachlysate pulsed onto antigen presenting cells for presentation tosyngeneic murine T cells both in vivo and in vitro. In a preferredembodiment, the tumors would derive by in vitro mutagenesis or oncogenetransformation from an immortalized, contact-inhibited,anchorage-dependent, non-tumorigenic cell line so that very well-matchednon-tumorigenic cells would be available for tolerance induction.

An alternative to the tolerization protocols is depletion of T cellsthat are activated by normal antigens prior to immunization with tumor.Activated T cells transiently express CD69 and CD25 with peak expressionbetween 24 and 48 hours post-stimulation. T cells expressing thesemarkers following activation with normal cells or normal cell lysatescan be depleted with anti-CD69 and anti-CD25 antibody coupled directlyor indirectly to a matrix such as magnetic beads. Subsequentimmunization of the remaining T cells with tumor cells or tumor celllysates either in vitro or in vivo following adoptive transfer willpreferentially give rise to a tumor-specific response.

In one embodiment, the mice to be tolerized to normal human cells orlysates and subsequently immunized with tumor cells or lysates are anyof a variety of commercially available inbred and outbred strains.Because murine T cells are restricted to recognize peptide antigens inassociation with murine MHC molecules which are not expressed by humancells, effective tolerization or stimulation requires eithertransfection of human cells with murine MHC molecules or re-presentationof human normal and tumor antigens by mouse antigen presenting cells.Dendritic cells are especially preferred as antigen presenting cellsbecause of their ability to re-present antigenic peptides in both theclass I and class II MHC pathways (Huang, et al., Science 264:961-965(1994); Inaba, et al., J. Exp. Med. 176:1702 (1992); Inaba, et al., J.Exp. Med. 178:479-488 (1993)). In another embodiment, mice doubletransgenic for human HLA and human CD8 or CD4 are employed. The HLAtransgene permits selection of a high affinity, HLA-restricted T cellrepertoire in the mouse thymus. In addition, a human CD8 or CD4transgene is required because murine CD8 and CD4 do not interactefficiently with the cognate human class I or class II MHC molecules.The use of non-transgenic mice to generate human tumor-specific T cellswould lead to identification of any human tumor antigens that can beprocessed in association with murine MHC molecules. Since multiplemurine strains with diverse MHC molecules are available, this couldencompass a wide range of antigens. However, it would have to beseparately determined by stimulation of human T cells with autologousantigen presenting cells whether these tumor-specific antigens alsoexpress peptides that can be processed and presented in association withhuman HLA. Such peptides may or may not overlap with those initiallydetected in association with murine MHC molecules but would derive fromthe same set of proteins. By employing HLA transgenic mice it ispossible to more directly address the relevance of antigenic peptides tohuman MHC. There can, however, be no assurance that peptide processingwill be identical in murine and human antigen presenting cells. It isessential, therefore, to confirm that HLA-restricted, human tumorantigen-specific T cells are indeed also crossreactive on human tumorcells. Finally, no matter how the issue of processing and presentationin association with human HLA is addressed, it must in all cases bedetermined whether human T cells are reactive to the identified antigensor whether they have been rendered tolerant, perhaps due to expressionof the same or a related antigen in some other non-homologous normaltissue. Relevant information on this point can be obtained through invitro stimulation of human T cell responses with the identified antigensor antigenic peptides presented by autologous antigen presenting cells.Ideally, it would be shown that patients with antigen positive tumorshave an increased frequency of T cells reactive with the purportedtumor-specific antigen. To demonstrate that the antigen-specific human Tcells induced can be effective in eradicating tumors, the selected humanT cells could be adoptively transferred into SCID mice bearing a humantumor xenograft as described by Renner, C. et al., Science 264:833-835(1994). However, definitive evidence for clinical relevance would awaitthe results of a human clinical trial.

Conditions for in vitro stimulation of primary human T cell responsesare described in Example 2 and are applicable to both CD4+ and CD8+responses. The strategies described for induction of human T cell orantibody responses specific for human tumors are equally applicable toinduction of T cell or antibody responses to target antigens of humancells infected with either a virus, fungus or mycobacteria. Indeed, inthis case the same uninfected cell population affords an immediatelyavailable normal control population for tolerance induction and toconfirm infectious specificity.

The construction of transgenic mice is well known in the art and isdescribed, for example, in Manipulating the Mouse Embroy: A laboratoryManual, Hogan, et al., Cold Spring Harbor Press, second edition (1994).Human CD8 transgenic mice may be constructed by the method of LaFace, etal., J. Exp. Med. 182:1315-25 (1995). Construction of new lines oftransgenic mice expressing the human CD8alpha and CD8beta subunits maybe made by insertion of the corresponding human cDNA into a human CD2minigene based vector for T cell-specific expression in transgenic mice(Zhumabekov, et al., J. Immunol. Methods 185:133-140 (1995)). HLA classI transgenic mice may be constructed by the methods of Chamberlain, etal., Proc. Natl. Acad. Sci. USA 85:7690-7694 (1988) or Bernhard, et al.,J. Exp. Med. 168:1157-1162 (1988) or Vitiello, et al., J. Exp. Med.173:1007-1015 (1991) or Barra, et al., J. Immunol. 150:3681-3689 (1993).

Construction of additional HLA class I transgenic mice may be achievedby construction of an H-2Kb cassette that includes 2 kb of upstreamregulatory region together with the first two introns previouslyimplicated in gene regulation (Kralova, et al., 1992, EMBO J. 11:4591-4600). Endogenous translational start sites are eliminated fromthis region and restriction sites for insertion of HLA cDNA areintroduced into the third exon followed by a polyA addition site. Byincluding an additional 3 kb of genomic H-2Kb sequence at the 3′ end ofthis construct, the class I gene can be targeted for homologousrecombination at the H-2Kb locus in embryonic stem cells. This has theadvantage that the transgene is likely to be expressed at a definedlocus known to be compatible with murine class I expression and thatthese mice are likely to be deficient for possible competition by H-2Kbexpression at the cell membrane. It is believed that this will giverelatively reproducible expression of diverse human HLA class I cDNAintroduced in the same construct.

Example 7 Construction of N-Terminal and/or C-Terminal Deletion Mutants

The following general approach may be used to clone a N-terminal orC-terminal deletion C35 deletion mutant. Generally, two oligonucleotideprimers of about 15-25 nucleotides are derived from the desired 5′ and3′ positions of a polynucleotide of SEQ ID NO:1. The 5′ and 3′ positionsof the primers are determined based on the desired C35 polynucleotidefragment. An initiation and stop codon are added to the 5′ and 3′primers respectively, if necessary, to express the C35 polypeptidefragment encoded by the polynucleotide fragment. Preferred C35polynucleotide fragments are those encoding the candidate MHC class Iand MHC class II binding peptides disclosed above in the “Polynucleotideand Polypeptide Fragments” section of the Specification.

Additional nucleotides containing restriction sites to facilitatecloning of the C35 polynucleotide fragment in a desired vector may alsobe added to the 5′ and 3′ primer sequences. The C35 polynucleotidefragment is amplified from genomic DNA or from the cDNA clone using theappropriate PCR oligonucleotide primers and conditions discussed hereinor known in the art. The C35 polypeptide fragments encoded by the C35polynucleotide fragments of the present invention may be expressed andpurified in the same general manner as the full length polypeptides,although routine modifications may be necessary due to the differencesin chemical and physical properties between a particular fragment andfull length polypeptide.

As a means of exemplifying but not limiting the present invention, thepolynucleotide encoding the C35 polypeptide fragment is amplified andcloned as follows: A 5′ primer is generated comprising a restrictionenzyme site followed by an initiation codon in frame with thepolynucleotide sequence encoding the N-terminal portion of an MHCbinding peptide epitope listed in any of Tables 1 through 6. Acomplementary 3′ primer is generated comprising a restriction enzymesite followed by a stop codon in frame with the polynucleotide sequenceencoding C-terminal portion of a C35 MHC binding peptide epitope listedin any of Tables 1 through 6.

The amplified polynucleotide fragment and the expression vector aredigested with restriction enzymes which recognize the sites in theprimers. The digested polynucleotides are then ligated together. The C35polynucleotide fragment is inserted into the restricted expressionvector, preferably in a manner which places the C35 polypeptide fragmentcoding region downstream from the promoter. The ligation mixture istransformed into competent E. coli cells using standard procedures andas described in the Examples herein. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA sequencing.

Example 8 Protein Fusions of C35

C35 polypeptides are preferably fused to other proteins. These fusionproteins can be used for a variety of applications. For example, fusionof C35 polypeptides to His-tag, HA-tag, protein A, IgG domains, andmaltose binding protein facilitates purification. (See Example 5; seealso EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).)Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflifetime in vivo. Nuclear localization signals fused to C35 polypeptides cantarget the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector.

For example, if pC4 (Accession No. 209646) is used, the human Fc portioncan be ligated into the BamHI cloning site. Note that the 3′ BamHI siteshould be destroyed. Next, the vector containing the human Fc portion isre-restricted with BamHI, linearizing the vector, and C35polynucleotide, isolated by the PCR protocol described in Example 1, isligated into this BamHI site. Note that the C35 polynucleotide is clonedwithout a stop codon, otherwise a fusion protein will not be produced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc Region:

(SEQ ID NO: [84]) GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

A preferred fusion product is fusion of a C35 peptide to the aminoterminus of an MHC molecule in such fashion that the peptide willnaturally occupy the MHC peptide binding groove. Kang, X. et al., CancerRes. 57:202-5 (1997) have reported that such fusion proteins can beemployed in vaccine compositions that are especially effective forstimulation of specific T cells.

C35 polypeptides can be detected in a biological sample, and if anincreased or decreased level of C35 is detected, this polypeptide is amarker for a particular phenotype. Methods of detection are numerous,and thus, it is understood that one skilled in the art can modify thefollowing assay to fit their particular needs.

Example 9 Method of Detecting Abnormal Levels of C35 in a BiologicalSample

For example, antibody-sandwich ELISAs are used to detect C35 in asample, preferably a biological sample. Wells of a microtiter plate arecoated with specific antibodies to C35, at a final concentration of 0.2to 10 μg/ml. The antibodies are either monoclonal or polyclonal. Thewells are blocked so that non-specific binding of C35 to the well isreduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining C35. Preferably, serial dilutions of the sample should beused to validate results. The plates are then washed three times withsaline to remove unbounded C35.

Next, 50 μl of specific antibody-alkaline phosphatase conjugate thatrecognizes a C35 antigenic determinant which does not overlap with thatrecognized by the plate bound antibody, at a concentration of 25-400 ng,is added and incubated for 2 hours at room temperature. The plates areagain washed three times with deionized or distilled water to removeunbounded conjugate.

Add 75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot C35 polypeptide concentration on the X-axis (log scale) andfluorescence or absorbance on the Y-axis (linear scale). Interpolate theconcentration of the C35 in the sample using the standard curve.

Example 10 Formulating a Polypeptide

The C35 composition will be formulated and dosed in a fashion consistentwith good medical practice, taking into account the clinical conditionof the individual patient (especially the side effects of treatment withthe C35 polypeptide alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofC35 administered parenterally per dose will be in the range of about 1μg/kg/day to 10 mg/kg/day of patient body weight, although, as notedabove, this will be subject to therapeutic discretion. More preferably,this dose is at least 0.01 mg/kg/day, and most preferably for humansbetween about 0.01 and 1 mg/kg/day. If given continuously, C35 istypically administered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

Pharmaceutical compositions containing C35 are administered orally,rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), bucally, or as an oral or nasal spray.“Pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

C35 is also suitably administered by sustained-release systems. Suitableexamples of sustained-release compositions include semi-permeablepolymer matrices in the form of shaped articles, e.g., films, ormirocapsules. Sustained-release matrices include polylactides (U.S. Pat.No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomally entrapped C35 polypeptides.Liposomes containing the C35 are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal secreted polypeptide therapy.

For parenteral administration, in one embodiment, C35 is formulatedgenerally by mixing it at the desired degree of purity, in a unit dosageinjectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptide.

Generally, the formulations are prepared by contacting C35 uniformly andintimately with liquid carriers or finely divided solid carriers orboth. Then, if necessary, the product is shaped into the desiredformulation. Preferably the carrier is a parenteral carrier, morepreferably a solution that is isotonic with the blood of the recipient.Examples of such carrier vehicles include water, saline, Ringer'ssolution, and dextrose solution. Non-aqueous vehicles such as fixed oilsand ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

C35 is typically formulated in such vehicles at a concentration of about0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8.It will be understood that the use of certain of the foregoingexcipients, carriers, or stabilizers will result in the formation ofpolypeptide salts.

C35 used for therapeutic administration can be sterile. Sterility isreadily accomplished by filtration through sterile filtration membranes(e.g., 0.2 micron membranes). Therapeutic polypeptide compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

C35 polypeptides ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous C35 polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized C35 polypeptide using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, C35 maybe employed in conjunction with other therapeutic compounds.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, Examples, and Sequence Listing is hereby incorporatedherein by reference.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, Examples, and Sequence Listing is hereby incorporatedherein by reference.

1. A method for treating or preventing a disorder in a mammalian subjectwhich comprises administering to said subject a therapeuticallyeffective amount of a pharmaceutical composition, wherein saidcomposition comprises one or more isolated C35 peptides consisting of anepitope selected from the group consisting of: amino acids S-9 to V-17of SEQ ID NO:2, amino acids V-10 to V-17 of SEQ ID NO:2, amino acidsE-16 to V-23 of SEQ ID NO:2, amino acids E-16 to R-24 of SEQ ID NO:2,amino acids E-16 to I-25 of SEQ ID NO:2, amino acids S-21 to F-35 of SEQID NO:2, amino acids C-30 to T-38 of SEQ ID NO:2, amino acids E-31 toY-39 of SEQ ID NO:2, amino acids E-36 to A-43 of SEQ ID NO:2, aminoacids A-37 to A-45 of SEQ ID NO:2, amino acids A-37 to V-46 of SEQ IDNO:2, amino acids Y-39 to V-46 of SEQ ID NO:2, amino acids S-44 to I-53of SEQ ID NO:2, amino acids A-45 to I-53; of SEQ ID NO:2, amino acidsG-52 to L-59 of SEQ ID NO:2, amino acids E-54 to T-62 of SEQ ID NO:2,amino acids S-57 to F-75 of SEQ ID NO:2, amino acids R-58 to I-67 of SEQID NO:2, amino acids G-61 to I-69 of SEQ ID NO:2, amino acids G-63 toF-83 of SEQ ID NO:2, amino acids E-66 to L-73 of SEQ ID NO:2, aminoacids E-66 to V-74 of SEQ ID NO:2, amino acids F-83 to E-103 of SEQ IDNO:2, amino acids D-88 to A-96 of SEQ ID NO:2, amino acids L-89 to A-96of SEQ ID NO:2, amino acids A-92 to T-101 of SEQ ID NO:2, amino acidsR-95 to L-102 of SEQ ID NO:2, amino acids A-96 to K-104 of SEQ ID NO:2,amino acids K-104 to V-113 of SEQ ID NO:2, amino acids I-105 to V-113 ofSEQ ID NO:2, and amino acids I-105 to I-114 of SEQ ID NO:2.
 2. Themethod of claim 1, wherein said disorder is a human carcinoma.
 3. Themethod of claim 2, wherein said carcinoma is selected from the groupconsisting of breast, bladder, ovarian, prostate, bone, liver, lung,pancreatic, and splenic carcinoma.
 4. The method of claim 3, whereinsaid carcinoma is breast carcinoma.
 5. The method of claim 3, whereinsaid carcinoma is bladder carcinoma.
 6. The method of claim 1, whereinsaid epitope is selected from the group consisting of: amino acids K-104to V-113 of SEQ ID NO:2, amino acids I-105 to V-113 of SEQ ID NO:2, andamino acids I-105 to I-114 of SEQ ID NO:2.
 7. The method of claim 1,wherein said composition further comprises an adjuvant.
 8. The method ofclaim 1, wherein said composition further comprises a pharmaceuticallyacceptable carrier.
 9. The method of claim 1, wherein said compositionis administered to said subject orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically, bucally,or as an oral or a nasal spray.
 10. The method of claim 9, whereinadministration to said subject parenterally.
 11. The method of claim 1,wherein said one or more C35 peptides are administered at a dose ofabout 1 μg/kg/day to about 10 mg/kg/day.
 12. The method of claim 11,wherein said one or more C35 peptides are administered at a dose ofabout 0.01 mg/kg/day to about 1 mg/kg/day.
 13. A method for generatingan immune response in an animal comprising administering a compositionto said animal, wherein said composition comprises one or more isolatedC35 peptides consisting of an epitope selected from the group consistingof: amino acids S-9 to V-17 of SEQ ID NO:2, amino acids V-10 to V-17 ofSEQ ID NO:2, amino acids E-16 to V-23 of SEQ ID NO:2, amino acids E-16to R-24 of SEQ ID NO:2, amino acids E-16 to I-25 of SEQ ID NO:2, aminoacids S-21 to F-35 of SEQ ID NO:2, amino acids C-30 to T-38 of SEQ IDNO:2, amino acids E-31 to Y-39 of SEQ ID NO:2, amino acids E-36 to A-43of SEQ ID NO:2, amino acids A-37 to A-45 of SEQ ID NO:2, amino acidsA-37 to V-46 of SEQ ID NO:2, amino acids Y-39 to V-46 of SEQ ID NO:2,amino acids S-44 to I-53 of SEQ ID NO:2, amino acids A-45 to I-53; ofSEQ ID NO:2, amino acids G-52 to L-59 of SEQ ID NO:2, amino acids E-54to T-62 of SEQ ID NO:2, amino acids S-57 to F-75 of SEQ ID NO:2, aminoacids R-58 to I-67 of SEQ ID NO:2, amino acids G-61 to I-69 of SEQ IDNO:2, amino acids G-63 to F-83 of SEQ ID NO:2, amino acids E-66 to L-73of SEQ ID NO:2, amino acids E-66 to V-74 of SEQ ID NO:2, amino acidsF-83 to E-103 of SEQ ID NO:2, amino acids D-88 to A-96 of SEQ ID NO:2,amino acids L-89 to A-96 of SEQ ID NO:2, amino acids A-92 to T-101 ofSEQ ID NO:2, amino acids R-95 to L-102 of SEQ ID NO:2, amino acids A-96to K-104 of SEQ ID NO:2, amino acids K-104 to V-113 of SEQ ID NO:2,amino acids I-105 to V-113 of SEQ ID NO:2, and amino acids I-105 toI-114 of SEQ ID NO:2.
 14. The method of claim 13, wherein said animal isselected from the group consisting of a mouse, a hamster, a dog, a cat,a monkey, a rabbit, a chimpanzee, and a human.
 15. The method of claim14, wherein said animal is human.
 16. The method of claim 13, furthercomprising administering to said animal a sufficient amount of said C35peptide to stimulate production of specific antibodies and/or T cells.17. The method of claim 16, wherein said antibodies and/or T-cells arespecific for a human carcinoma.
 18. The method of claim 17, wherein saidcarcinoma is selected from the group consisting of breast, bladder,ovarian, prostate, bone, liver, lung, pancreatic, and splenic carcinoma.19. The method of claim 18, wherein said carcinoma is breast carcinomaor bladder carcinoma.
 20. The method of claim 13, wherein said epitopeis selected from the group consisting of: amino acids K-104 to V-113 ofSEQ ID NO:2, amino acids I-105 to V-113 of SEQ ID NO:2, and amino acidsI-105 to I-114 of SEQ ID NO:2.